Multimeric IL-15 soluble fusion molecules and methods of making and using same

ABSTRACT

The present invention features compositions and methods featuring ALT-803, a complex of an interleukin-15 (IL-15) superagonist mutant and a dimeric IL-15 receptor α/Fc fusion protein useful for enhancing an immune response against a neoplasia (e.g., multiple myeloma, melanoma, lymphoma) or a viral infection (e.g., human immunodeficiency virus).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/951,042, filed Apr. 11, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/083,998, filed Mar. 29, 2016, which is acontinuation of U.S. patent application Ser. No. 13/854,903, filed Apr.1, 2013, which is a continuation of U.S. patent application Ser. No.13/769,179, filed Feb. 15, 2013, which is a continuation-in-part of U.S.patent application Ser. No. 13/238,925, filed Sep. 21, 2011, whichclaims the benefit of U.S. Provisional Patent Application Ser. No.61/527,911, filed Aug. 26, 2011 and claims the benefit of U.S.Provisional Patent Application No. 61/384,817, filed Sep. 21, 2010. U.S.patent application Ser. No. 13/854,903, filed Apr. 1, 2013, is also acontinuation-in-part of said U.S. patent application Ser. No.13/238,925. The entire contents of each of the foregoing applicationsare hereby incorporated by reference in their entireties.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The contents of the text file named “48277_521C05.txt”, which wascreated on Jan. 21, 2014 and is 68 KB in size, is hereby incorporated byreference in its entirety and for all purposes.

BACKGROUND OF THE INVENTION

Multiple myeloma (MM) is a plasma cell malignancy, accounting for over1% of neoplastic diseases and 14% of all hematological cancers. MM tumorcells are susceptible to immune cell recognition and elimination, asdemonstrated by the potentially curative graft-versus-myeloma activityobserved in some patients following allogeneic hematopoietic stem celltransplantation and donor lymphocyte infusion therapies. However, theseapproaches are limited by transplantation-related mortality ranging from30% to 50% and disease relapse in a majority of patients.Immunomodulatory chemotherapies, such as lenalidomide, are also thoughtto provide therapeutic benefit via mechanisms due in part to stimulationof T-cell and/or natural killer (NK) cell activity against myelomacells. Although survival of MM patients has improved significantly bythe use of these novel agents, MM remains incurable due to thepersistence of minimal residual disease. Thus, novel modalities areneeded to complement or improve the current treatment options for MM.

SUMMARY OF THE INVENTION

As described below, the present invention features compositions andmethods featuring ALT-803, a complex of an interleukin-15 (IL-15)superagonist mutant and a dimeric IL-15 receptor α/Fc fusion proteinuseful for enhancing an immune response against a neoplasia (e.g., ahematological cancer, multiple myeloma, beta-cell lymphoma,urothelial/bladder carcinoma and melanoma) or a viral infection (e.g.,human immunodeficiency virus).

In one aspect, the invention features a method for treating neoplasia orvirus infection in a subject (e.g., human), the method containingadministering to the subject an effective amount of a pharmaceuticalcomposition containing IL-15N72D:IL-15RαSu/Fc complex (Alt-803)containing a dimeric IL-15RαSu/Fc and two IL-15N72D molecules, therebytreating the neoplasia or virus infection. In one embodiment, theIL-15RαSu/Fc comprises the following sequences (“IL-15RαSu/Fc” disclosedas SEQ ID NO: 1):

itcpppmsvehadiwvksyslysreryicnsgfkrkagtssltecvlnkat nvahwttpslkcir-[IL-15RαSu] epkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk [IgG1 CH2—CH3 (Fc domain)].

In another embodiment, the IL-15N72D molecule comprises the followingsequence (SEQ ID NO: 2):

nwvnvisdlkkiedliqsmhidatlytesdvhpsckvtamkcfllelqvislesgdasihdtvenliilandslssngnvtesgckeceeleeknikeflqs fvhivqmfints[IL-15N72D].

In another aspect, the invention features a kit for the treatment of aneoplasia, the kit containing an effective amount of anIL-15N72D:IL-15RαSu/Fc complex (Alt-803) containing a dimericIL-15RαSu/Fc and two IL-15N72D molecules and directions for the use ofthe kit for the treatment of a neoplasia.

In another aspect, the invention features a kit for the treatment of avirus (e.g., HIV), the kit containing an effective amount of anIL-15N72D:IL-15RαSu/Fc complex (Alt-803) containing a dimericIL-15RαSu/Fc and two IL-15N72D molecules and directions for the use ofthe kit for the treatment of a neoplasia.

In another aspect, the invention features a method of treating neoplasiain a subject, the method containing administering to said subject aneffective amount of a pharmaceutical composition containing an anti-CD20scAb T2M complex or a CD20-targeted IL-15N72D:IL-15Rα/Fc fusion proteincomplex (2B8T2M), thereby treating the neoplasia. In one embodiment, theanti-CD20 scAb T2M contains a soluble anti-CD20scAb/huIL-15N72D:anti-CD20 scAb/huIL-15RαSu/huIgG1 Fc complex, whereinanti-CD20 scAb/huIL-15RαSu/huIgG1 Fc has the sequence shown in FIG. 56,and the anti-CD20 scAb/hIL-15N72D has the sequence shown in FIG. 54. Inanother embodiment, the neoplasia is beta-cell lymphoma or Burkitt'slymphoma. In another embodiment, the effective amount is between about 1and 100 μg/kg. In yet another embodiment, Alt-803 is administered once,twice, or three times per week. In various embodiments of the aboveaspects or any other aspect of the invention delineated herein, theneoplasia is multiple myeloma, beta-cell lymphoma, urothelial/bladdercarcinoma or melanoma. In other embodiments, the effective amount isbetween about 1 and 20 μg/kg. In other embodiments, the effective amountis about 1 μg/kg, 5 μg/kg, 10 μg/kg, 15 μg/kg, or 20 μg/kg. In stillother embodiments, the effective amount is 10 μg/week. In still otherembodiments, the effective amount is between about 20 μg and 100 μg/kg.In still other embodiments, the effective amount is 30 μg/kg, 50 μg/kg,75 μg/kg, or 100 μg/kg. In still other embodiments, Alt-803 isadministered once, twice, or three times per week. In yet otherembodiments, the pharmaceutical composition is administeredsystemically, intravenously, or by instillation. In still otherembodiments, Alt-803 increases serum levels of IFN-γ; increases thenumber of CD8⁺CD44^(high) memory T cells, causes CD8⁺CD44^(high) memoryT cells to acquire an innate-type phenotype and secrete IFN-γindependent of antigen requirement; and/or induces a long lastinganti-myeloma immune memory response. In still other embodiments, thevirus is human immunodeficiency virus.

The invention provides therapeutic compositions useful for enhancing animmune response and methods of using such compositions for the treatmentof viral infections, such as HIV, and neoplasias, including but notlimited to multiple myeloma, beta-cell lymphoma, urothelial/bladdercarcinoma and melanoma. Compositions and articles defined by theinvention were isolated or otherwise manufactured in connection with theexamples provided below. Other features and advantages of the inventionwill be apparent from the detailed description, and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “agent” is meant a peptide, nucleic acid molecule, or small compound.An exemplary therapeutic agent is Alt-803 or 2B8T2M.

By “Alt-803” is meant a complex comprising IL-15N72D noncovalentlyassociated with a dimeric IL-15RαSu/Fc fusion protein and having immunestimulating activity. In one embodiment, the IL-15N72D and/orIL-15RαSu/Fc fusion protein comprises one, two, three, four or moreamino acid variations relative to a reference sequence. An exemplaryIL-15N72D amino acid sequence is provided below.

IL-15N72D protein sequence (with leader peptide) (SEQ ID NO: 3)metdtlllwvlllwvpgstg- [Leader peptide]nwvnvisdlkkiedliqsmhidatlytesdvhpsckvtamkcfllelqvislesgdasihdtvenliilandslssngnvtesgckeceeleeknikeflqs fvhivqmfints[IL-15N72D]In one embodiment, the leader peptide is cleaved from the matureIL-15N72D polypeptide.

An exemplary IL-15RαSu/Fc amino acid sequence is provided below:

IL-15RαSu/Fc protein sequence (with leader peptide) (SEQ ID NO: 4)mdrltssflllivpayvls- [Leader peptide]itcpppmsvehadiwvksyslysreryicnsgfkrkagtssltecvlnkat nvahwttpslkcir-[IL-15RαSu] epkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk [IgG1 CH2—CH3 (Fc domain)]

In one embodiment, the mature IL-15RαSu/Fc protein lacks the leadersequence. Other Alt-803 polypeptide and polynucleotide sequences usefulin the method of the invention are provided at FIG. 86A, FIG. 86B, andFIG. 86C.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

By “analog” is meant a molecule that is not identical, but has analogousfunctional or structural features. For example, a polypeptide analogretains the biological activity of a corresponding naturally-occurringpolypeptide, while having certain biochemical modifications that enhancethe analog's function relative to a naturally occurring polypeptide.Such biochemical modifications could increase the analog's proteaseresistance, membrane permeability, or half-life, without altering, forexample, ligand binding. An analog may include an unnatural amino acid.

By “binding to” a molecule is meant having a physicochemical affinityfor that molecule.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

“Detect” refers to identifying the presence, absence or amount of theanalyte to be detected.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.Examples of diseases include neoplasias and viral infections.

By “effective amount” is meant the amount of a required to amelioratethe symptoms of a disease relative to an untreated patient. Theeffective amount of active compound(s) used to practice the presentinvention for therapeutic treatment of a disease varies depending uponthe manner of administration, the age, body weight, and general healthof the subject. Ultimately, the attending physician or veterinarian willdecide the appropriate amount and dosage regimen. Such amount isreferred to as an “effective” amount.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 nucleotides or amino acids.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. “Isolate” denotes a degree ofseparation from original source or surroundings. “Purify” denotes adegree of separation that is higher than isolation. A “purified” or“biologically pure” protein is sufficiently free of other materials suchthat any impurities do not materially affect the biological propertiesof the protein or cause other adverse consequences. That is, a nucleicacid or peptide of this invention is purified if it is substantiallyfree of cellular material, viral material, or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Purity and homogeneity aretypically determined using analytical chemistry techniques, for example,polyacrylamide gel electrophoresis or high performance liquidchromatography. The term “purified” can denote that a nucleic acid orprotein gives rise to essentially one band in an electrophoretic gel.For a protein that can be subjected to modifications, for example,phosphorylation or glycosylation, different modifications may give riseto different isolated proteins, which can be separately purified.

By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) thatis free of the genes which, in the naturally-occurring genome of theorganism from which the nucleic acid molecule of the invention isderived, flank the gene. The term therefore includes, for example, arecombinant DNA that is incorporated into a vector; into an autonomouslyreplicating plasmid or virus; or into the genomic DNA of a prokaryote oreukaryote; or that exists as a separate molecule (for example, a cDNA ora genomic or cDNA fragment produced by PCR or restriction endonucleasedigestion) independent of other sequences. In addition, the termincludes an RNA molecule that is transcribed from a DNA molecule, aswell as a recombinant DNA that is part of a hybrid gene encodingadditional polypeptide sequence.

By an “isolated polypeptide” is meant a polypeptide of the inventionthat has been separated from components that naturally accompany it.Typically, the polypeptide is isolated when it is at least 60%, byweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight, a polypeptide of the invention. An isolated polypeptideof the invention may be obtained, for example, by extraction from anatural source, by expression of a recombinant nucleic acid encodingsuch a polypeptide; or by chemically synthesizing the protein. Puritycan be measured by any appropriate method, for example, columnchromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By “marker” is meant any protein or polynucleotide having an alterationin expression level or activity that is associated with a disease ordisorder.

By “neoplasia” is meant a disease or disorder characterized by excessproliferation or reduced apoptosis. Illustrative neoplasms for which theinvention can be used include, but are not limited to leukemias (e.g.,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia,acute myeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease,non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chaindisease, and solid tumors such as sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, glioblastomamultiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma,schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). Inparticular embodiments, the neoplasia is multiple myeloma, beta-celllymphoma, urothelial/bladder carcinoma or melanoma. As used herein,“obtaining” as in “obtaining an agent” includes synthesizing,purchasing, or otherwise acquiring the agent.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%,75%, or 100%.

By “reference” is meant a standard or control condition.

A “reference sequence” is a defined sequence used as a basis forsequence comparison. A reference sequence may be a subset of or theentirety of a specified sequence; for example, a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence. For polypeptides, the length of the reference polypeptidesequence will generally be at least about 16 amino acids, preferably atleast about 20 amino acids, more preferably at least about 25 aminoacids, and even more preferably about 35 amino acids, about 50 aminoacids, or about 100 amino acids. For nucleic acids, the length of thereference nucleic acid sequence will generally be at least about 50nucleotides, preferably at least about 60 nucleotides, more preferablyat least about 75 nucleotides, and even more preferably about 100nucleotides or about 300 nucleotides or any integer thereabout ortherebetween.

By “specifically binds” is meant a compound or antibody that recognizesand binds a polypeptide of the invention, but which does notsubstantially recognize and bind other molecules in a sample, forexample, a biological sample, which naturally includes a polypeptide ofthe invention.

Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule.Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary polynucleotide sequences (e.g., a gene described herein),or portions thereof, under various conditions of stringency. (See, e.g.,Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less thanabout 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and more preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and more preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred: embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100.mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferredembodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mMtrisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Usefulvariations on these conditions will be readily apparent to those skilledin the art.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a more preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows fusion protein referred to as the T2 molecule (T2M)consists of a multichain polypeptide.

FIG. 2 shows the vector (pMC.c264scTCR-Su/IgG1.PUR) containing the humanIL15RαSushi gene insert.

FIG. 3A, FIG. 3B, and FIG. 3C shows the sequence of thec264scTCR/huIL15RαSushi/huIgG1 nucleic acid sequence (SEQ ID NO: 39).

FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D shows the protein sequence of thec264scTCR/huIL15RαSushi/huIgG1 peptide (SEQ ID NO: 40).

FIG. 5 shows the vector designated as c264scTCR/Sushi/hIgG1-pMSGVc orpMSGVc264SuIg.

FIG. 6A, FIG. 6B, and FIG. 6C shows the sequence of thec264scTCR/huIL15RαSushi/huIgG1 nucleic acid sequence (SEQ ID NO: 41).

FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D shows the protein sequence of thec264scTCR/huIL15RαSushi/huIgG1 peptide (SEQ ID NO: 42).

FIG. 8 shows the vector designated as c149scTCR/IL15N72D-pMSGVn orpMSGV-c149IL15N72D.

FIG. 9A and FIG. 9B shows the sequence of the c149scTCR/huIL15N72Dnucleic acid sequence (SEQ ID NO: 43).

FIG. 10A, FIG. 10B, and FIG. 10C shows the protein sequence of thec149scTCR/huIL15N72D peptide (SEQ ID NO: 44).

FIG. 11 shows an SDS-PAGE analysis of purification fractions of the T2,c264scTCR/huIgG1 and c264scTCR/huIgG1ΔCH1 fusion proteins under reducingand non-reducing conditions. Under reducing conditions, the T2 moleculebands migrate at molecular weights consisted with the c264scTCR/huIL15and c264scTCR/huIL15RαSushi/huIgG1 polypeptides. Under non-reducingdenaturing conditions, the c264scTCR/huIL15RαSushi/huIgG1 band migratesat a molecular weight consistent with a dimeric disulfide-linkedc264scTCR/huIL15RαSushi/huIgG1 complex and a c264scTCR/huIL15N72Dpolypeptide.

FIG. 12 shows results from size exclusion gel filtration chromatographydemonstrating that the native T2 protein eluted at the expectedmolecular weight of a four-chain (2×c264scTCR/IL15N72D,2×c264scTCR/huIL15RαSushi/huIgG1) molecule.

FIG. 13 shows results from an in vitro binding assay in which equimolaramounts of purified T2 protein, composed of c264scTCR/huIL15N72D andc264scTCR/huIL15RαSushi/huIgG1 chains, or purified c264scTCR/huIgG1fusion protein were captured on wells coated with anti-human IgG1antibody. Following binding, proteins were detected using anti-humanIgG1 antibody under standard ELISA conditions.

FIG. 14 shows results from an in vitro binding assay in which equimolaramounts of T2 or c264scTCR/huIgG1 proteins were captured on anti-humanIgG1 Ab coated wells and detected with an anti-human TCR Cβ antibody(W4F).

FIG. 15 shows results from an in vitro binding assay in which thepeptide/MHC binding activity of the TCR domains of the T2 molecule wasassessed. Equimolar amounts of T2 (composed of c264scTCR/huIL15N72D andc264scTCR/huIL15RαSushi/huIgG1 chains) or c264scTCR/huIgG1 proteins werecaptured on anti-human IgG1 Ab coated wells and detected with p53 (aa264-272) peptide/HLA-A2 streptavidin-HRP tetramers.

FIG. 16 shows results from an in vitro assay to demonstrate the activityof the IL-15 domain of the T2 molecule. Microtiter wells were coatedwith anti-human IL-15 antibody and equivalent molar amounts of purifiedT2 protein, composed of c264scTCR/huIL15N72D andc264scTCR/huIL15RαSushi/huIgG1 chains, or purified c264scTCR/huIL15N72Dfusion protein were applied to the wells. Following binding and washingsteps, the bound proteins were detected with anti-human IL-15 antibodyunder standard ELISA conditions.

FIG. 17 shows the results from a proliferation assay to furthercharacterize the functional activity of the IL-15 domain of the T2molecules using the cytokine-dependent 32Dβ cell line. To measure cellproliferation, 32Dβcells (2×10⁴ cells/well) were incubated withincreasing concentrations of T2 protein (composed ofc264scTCR/huIL15N72D and c264scTCR/huIL15RαSushi/huIgG1 chains) orc264scTCR/huIL15N72D fusion protein for 48 h at 37° C. Cellproliferation reagent WST-1 (Roche® Applied Science) was added duringthe last 4 h of cell growth according to the manufacturer's procedures.Conversion of WST-1 to the colored formazan dye by metabolically activecells was determined through absorbance measurements at 440 nm.

FIG. 18A and FIG. 18B show the results from an in vivo primate model todetermine the ability of the T2 protein to promote proliferation ofIL-15 responsive immune cells. Blood was collected five days afterinjection with T2 protein and was stained for CD8 memory T cells markers(CD8 and CD95) (FIG. 18A) and NK cell markers (CD56 and CD16) (FIG. 18B)and compared to blood taken prior to treatment.

FIG. 19A and FIG. 19B show cell binding assays characterizing thebinding activity of the IgG1 Fc domain of the T2 molecule. FIG. 19Aillustrates flow cytometry analysis showing results from an assay inwhich Fc-gamma receptor bearing U937 cells were incubated with 33 nM ofT2 protein (composed of c264scTCR/huIL15N72D andc264scTCR/huIL15RαSushi/huIgG1 chains), c264scTCR/huIgG1 orA2AL9scTCR/IgG1 (negative control) for 20 min. Cells were washed onceand incubated with PE-conjugated p53 (aa 264-272) peptide/HLA-A2tetramer for 20 min. The binding to Fc gamma receptors on U937 cellsurface was analyzed with flow cytometry. FIG. 19B illustrates flowcytometry analysis showing results from similar U937 binding studiesusing a range of protein concentrations as indicated. The meanfluorescent intensity for the stained cells was plotted.

FIG. 20 shows results from an assay to assess the biological activity ofthe Fc domains of the T2 molecules to mediate antibody dependentcellular cytotoxicity activity. T2 protein, c264scTCR/huIgG1 orA2AL9scTCR/IgG1 (negative control) were added to a 96-well plate at aconcentration of 0.137 nM to 100 nM. HLA-A2-positive T2 target cellswere pulsed with 10 μM of p53 aa264-272 peptide and labeled with 50μg/ml of Calcein-AM. The fusion proteins were mixed with 1×10⁴ of thetarget cell per well and 1×10⁶/well of fresh human PBMC were added. Theplate was incubated at 37° C. in a CO₂ incubator for 2 hrs and 100 μl ofthe conditional medium were collected and analyzed quantitatively forCalcein released from lysed cells.

FIG. 21A and FIG. 21B show results from an assay in whichHLA-A2-positive T2 cells were pulsed with various amounts of p53aa264-272 peptide to assess the binding activity of T2 protein topeptide/MHC targets on cell surface. The peptide-loaded cells wereincubated with T2 protein (composed of c264scTCR/huIL15N72D andc264scTCR/huIL15RαSushi/huIgG1 chains), c264scTCR/huIgG1 orA2AL9scTCR/IgG1 (negative control), each at 83 nM. The cells wereincubated with biotinylated anti-TCR Ab (BF1) and streptavidin-PE. Thecells were then analyzed for antibody staining by flow cytometry (FIG.21A) and the mean fluorescence staining intensity of the cells loadeddifferent concentrations of peptide are plotted (FIG. 21B).

FIG. 22 shows the results from an ELISA in which T2 molecules ofc149scTCR/huIL15N72D and c264scTCR/huIL15RαSushi/huIgG1 orc264scTCR/huIL15N72D and c264scTCR/huIL15RαSushi/huIgG1 (in cell culturesupernatant) were captured on microtiter plates coated with theanti-human TCR antibody BF1, and the bound T2 molecules were detectedusing the anti-human TCR antibody W4F-BN.

FIG. 23 shows the results from an ELISA in which T2 molecules ofc149scTCR/huIL15N72D and c264scTCR/huIL15RαSushi/huIgG1 orc264scTCR/huIL15N72D and c264scTCR/huIL15RαSushi/huIgG1 (in cell culturesupernatant) were captured on microtiter plates coated with the goatanti-human IgG antibody, and bound T2 molecules were detected using theanti-human IL-15 antibody.

FIG. 24 shows the results from an ELISA in which T2 molecules ofc149scTCR/huIL15N72D and c264scTCR/huIL15RαSushi/huIgG1 (in cell culturesupernatant) were captured on microtiter plates were coated with eithergoat anti-human IgG antibody or anti-human TCR antibody BF1. TheBF1-captured T2 molecules were detected with either anti-human TCRantibody W4F-BN or anti-human IL-15 antibody. The goat anti-human IgGAb-captured T2 molecules were detected with either the p53 (aa 149-157)peptide/HLA-A2 streptavidin-HRP tetramers or the p53 (aa 264-272)peptide/HLA-A2 streptavidin-HRP tetramers.

FIG. 25 shows results from a flow cytometry assay in which T2 moleculescomprising two different TCR domains, i.e. c264scTCR/huIL15N72D andc149scTCR/huIL15RαSushi/huIgG1 chains, were characterized. The Fc andTCR activity of these molecules were assessed following binding toFc-gamma receptor bearing U937 cells and detection with p53 (aa 264-272)peptide/HLA-A2 tetramers followed by flow cytometry.

FIG. 26A and FIG. 26B show the results from a pharmacokinetic assay inwhich mice (FIG. 26A) or monkeys (FIG. 26B) were injected with purifiedT2 protein composed of c264scTCR/huIL15N72D andc264scTCR/huIL15RαSushi/huIgG1 chains. Samples were collected at theindicated times. FIG. 26A is a line graph showing the results of ELISAformat assays in which goat anti-human IgG Ab was used to coat thewells, and anti-human TCR Ab (W4F-BN) was used for detection; or goatanti-human IgG Ab was used to coat the plates, and anti-human IL-15 Abwas used for detection as indicated to quantify the amount of the T2protein in the blood at the times indicated. FIG. 26B is a line graphshowing the results of assays in which anti-human TCR Ab (βF-1) was usedto coat the wells, and HRP conjugated goat anti-human IgG Ab was usedfor detection; or anti-human IL-15 Ab was used to coat the plates, andHRP conjugated goat anti-human IgG Ab was used for detection; oranti-human IL-15 Ab was used to coat the plates and anti-human TCR Ab(W4F-BN) was used for detection.

FIG. 27 shows results from a primary tumor growth model using a humanp53+ HLA-A2+ A375 melanoma cell line in nude mice. Tumor-bearing micewere injected intravenously with 32 μg/dose (1.6 mg/kg) T2 proteincomposed of c264scTCR/huIL15N72D and c264scTCR/huIL15RαSushi/huIgG1chains, 32 μm/dose (1.6 mg/kg) c264scTCR/huIL2, or 60 μg/dose (3 mg/kg)264scTCR/huIgG1. Tumor growth was measured and data are shown in thefigure.

FIG. 28 shows the results from IL-15 activity assays of T2 moleculeswith various point mutations in the IL-15 domain as measured byproliferation of 32Dβ cells.

FIG. 29 shows results from an antibody dependent cellular cytotoxicityassay using T2 molecules with various point mutations in the IL-15 andIgG Fc domains as measured by PBMC-dependent lysis of peptide-loaded T2target cells.

FIG. 30 shows results from an assay to detect the effects of the IL-15and Fc mutations on the ability of the T2 molecules to stimulate humanNK and T cell responses. Human PBMCs at 1.8 to 5×10⁵ cells/mL wereincubated for 4 days at 37° C. in media containing 1 nM T2 moleculescomprising the mutations indicated or with 10 ng/mL recombinant humanIL-2 or IL-15 as a control. NK cell cytotoxicity was then assessed usingNK-sensitive K-562 cells as target cells following labeling with 50ug/ml of Calcein-AM.

FIG. 31 shows results from NK cell proliferation assay in which humanPBMCs were incubated with T2 molecules comprising various pointmutations in the IL-15 and IgG Fc domains or with recombinant human IL-2or IL-15 as a control. T2 molecules comprising thec264scTCR/huIL15RαSushi/huIgG1 and c264scTCR/huIL15N72D chains or thosewith the Fc domain LALA and KA variants resulted in an increase inproliferation of CD56+ NK cells whereas T2 molecules comprising IL-15N65D or D8N substitutions did not provide as much NK cell proliferativeactivity.

FIG. 32A and FIG. 32B show results from flow cytometry assays to testthe antigen specific binding of T2 molecules including IL-15 and Fcmutations toT2 cells with (T2.265) and without loaded p53 peptide (T2).FIG. 32A shows flow cytometry histograms and FIG. 32B shows signal tonoise ratio of peptide-specific to non-specific cell staining.

FIG. 33A, FIG. 33B, and FIG. 33C show results from assays to detect theactivity of various T2 molecules and IL-15 molecules to support 32Dβcell growth (FIG. 33A) to stimulate expansion of various T cellpopulations (FIG. 33B), and to stimulate NK cell activity (FIG. 33C).

FIG. 34A and FIG. 34B show results from an in vivo assay to determinethe immunostimulatory activity of various T2 molecules in mice asindicated by changes in the percentage of CD8+ T-cells (FIG. 34A) and NKcells (FIG. 34B) in blood and spleen cells as detected using flowcytometry.

FIG. 35A and FIG. 35B show results from an ELISA using a multispecificT2 molecule comprising 1) the huIL15N72D domain fused to a scTCRspecific to the peptide from amino acids 257-264 of ovalbumin and 2) asingle chain CD8α/β domain linked to the huIL15RαSushi/huIgG1 fusion.Binding activity of OT1-CD8-T2M was compared to that of theOT1scTCR/huIL15N72D fusion by ELISA. Equal molar amounts of each proteinwas captured on a well coated with anti-TCR Cβ mAb (H57) and probed withOVA aa257-264/H-2Kb tetramers (FIG. 35B) or mAbs to IL15, CD8α, CD8β orTCR Vα2 (FIG. 35A). Assays were also performed with wells coated withanti-human Ig and probed with anti-TCR Vα2.

FIG. 36A shows a schematic diagram of thec264scTCR/hIL-15:c264scTCR/hIL-15RαSu/birA complex (c264scTCR dimer).The model of the dimeric hIL-15:hIL-15RαSu domains is based on thepublished crystal structure of the human IL-15:IL-15Rα complex (33) (PDB2Z3Q) FIG. 36B shows SEC analysis of c264scTCR fusion proteins. Panelsshow size analysis of c264scTCR/hIL-15 (top), c264scTCR/hIL-5RαSu/birA(middle) and c264scTCR/hIL-15:c264scTCR/hIL-15RαSu/birA complex(c264scTCR dimer) (bottom) with dashed lines indicating relative proteinpeaks.

FIG. 37A-FIG. 37C show characterization of the binding activity of thec264scTCR dimer comprising thec264scTCR/hIL-15:c264scTCR/hIL-15RαSu/birA complex andc264scTCR/c149scTCR heterodimer comprising thec149scTCR/hIL-15:c264scTCR/hIL-15RαSu/birA complex. T2 cells were pulsedwith 0-62.5 nM of p53 (aa264-272) peptide. The cells were stained withequivalent amounts (80 nM) of PE-conjugated multimers of the c264scTCRdimer or c264scTCR/birA (FIG. 37A). The relative increase in cellstaining comparing c264scTCR dimer with c264scTCR/birA reagents wasdetermined at different peptide concentrations (FIG. 37B). Foldincrease=(Geo mean of T2 cells stained by c264scTCR dimer)/(Geo Mean ofT2 cells stained by c264scTCR/birA). The p53 peptide/HLA-A*0201 bindingactivity of c264scTCR/c149scTCR heterodimer was determined by ELISA(FIG. 37C). Anti-hIL-15 monoclonal antibody (R&D System) was used as acapturing antibody. A2/p53.264-272.HRP or A2/p53.149-157.HRP tetramerswere used as the probes. The data represent the means±SD of triplicatedeterminations.

FIG. 38 shows the characterization of the binding activity of theOT1scTCR dimer comprising the OT1scTCR/hIL-15:OT1scTCR/hIL-15RαSu/birAcomplex. EL4 cells were loaded with OVA (aa257-264) peptide and stainedwith OT1scTCR/birA-SA-PE (top) and OT1scTCR dimer-SA-PE (bottom) at 200nM.

FIG. 39A-FIG. 39B shows OTscTCR/scCD8 heterodimer comprising theOT1scTCR/hIL-15:scCD8/hIL-15RαSu/birA complex exhibits enhanced pMHCIbinding activity. Murine CD8 expression of OT1scTCR/scCD8 heterodimerwas determined by ELISA (FIG. 39A). Anti-mTCR H57-597 mAb was used ascapturing antibody. The biotinylated anti-murine CD8α or CD8β mAb wasused as a probe followed by SA-HRP. The data represent the means±SD oftriplicate determinations. EL4 cells were loaded with OVA (aa257-264)peptide at the indicated concentration and stained with OT1scTCRdimer-SA-PE (top) and OT1scTCR/scCD8 heterodimer-SA-PE (bottom) at 200Nm (FIG. 39B).

FIG. 40A-FIG. 40B show that fusion proteins containing TCR α/βheterodimers comprising the TCRα/hIL-15:TCRβ/hIL-15RαSu/birA complexretain pMHCI binding activity. Binding activity of OT1scTCR/birA and OT1TCRα/β heterodimer to OVA (aa257-264)/H-2Kb complex was determined byELISA (FIG. 40A). Anti-mTCR H57-597 mAb was used as capturing antibody.Kb/OVA.257-264.HRP tetramer was used as a probe. Binding activity of264scTCR/birA and 264 TCRα/β heterodimer to p53 (aa264-272)/HLA-A*0201complex was determined by ELISA (FIG. 40B). Anti-TCR mAb was used ascapturing antibody. A2/p53.264-272.HRP tetramer was used as a probe. Thedata represent the means±SD of triplicate determinations.

FIG. 41A and FIG. 41B shows IL-15 binding and functional activity offusion proteins. 32Dβ cells were incubated with 320 nM of the c264scTCRdimers comprising IL-15 wild type or IL-15N72D or IL-15D8N muteindomains. The binding of the fusion proteins was in turn detected withanti-human TCR Cβ Ab (FIG. 41A). The ability of the c264scTCR dimerscomprising IL-15 wild type or mutein domains to support proliferation of32Dβ cells was determined as described in the Examples (FIG. 41B). Thedata represent the means±range of duplicate determinations.

FIG. 42 shows OVA (aa257-264)/H-2K^(b) binding activity ofOT1scTCR/hIL-15D8N, OT1scTCR/hIL-15RαSu/birA and OT1scTCR dimer weredetermined by ELISA. Anti-mTCR H57-597 mAb was used as capturingantibody. Kb/OVA.257-264.HRP tetramer was used as a probe. The datarepresent the means±SD of triplicate determinations.

FIG. 43 shows OT1scTCR fusion protein binding curves to OVA(aa257-264)/H-2K^(b) and control VSV/H-2K^(b) complexes determined bySPR.

FIG. 44A and FIG. 44B shows results from a primary tumor growth modelusing murine B16 melanoma tumor cell line in immunocompetent mice.Tumor-bearing mice were injected intravenously with rhlL-15, T2M,T2MΔCH1 and T2MΔTCRΔCH1 (Alt-803) proteins or PBS (control). Tumorgrowth was measured and data are shown in FIG. 44A. Post treatmentchanges in animal body weight are shown in FIG. 44B. These results showthat Alt-803 is effective for treating melanoma.

FIG. 45A and FIG. 45B show results from a primary tumor growth modelusing a murine EG7 thymoma/lymphoma tumor cell line in immunocompetentmice. Tumor-bearing mice were injected intravenously with rhlL-15, T2Mand T2MΔTCRΔCH1 (Alt-803) proteins or PBS (control). Tumor growth wasmeasured and data are shown in FIG. 45A. Post treatment changes inanimal body weight are shown in FIG. 45B. These data demonstrate thatAlt-803 is effective against thymoma/lymphoma.

FIG. 46 shows the protein sequence of the human IgG1 CH2-CH3 domain orFc domain covalently and/or genetically fused with other protein domainsto generate the fusion protein complexes (SEQ ID NO: 45).

FIG. 47 shows results of an assay to determine the antibody dependentcellular cytotoxicity activity mediated by T2M and scTCR-huIgG1 proteinsagainst cells expressing peptide MHC targets. Various amounts of fusionprotein (T2M, T2M2 or c264scTCR-Ig) were mixed with fresh human PBMCsand p53 peptide-pulsed HLA-A2-positive T2 cells (Calcein labeled) (E:Tratio, 40:1). After 2 hr incubation, the culture medium was collectedand analyzed quantitatively for Calcein released from lysed cells.

FIG. 48A-FIG. 48C shows results from in vivo assays to determine theimmunostimulatory activity of various T2 molecules in mice. C57BL/6 micewere treated i.v. with equivalent molar IL-15 doses of hIL-15 (1 mg/kg),IL15N72D:IL15Rα-Fc (3.6 mg/kg), T2M (11 mg/kg), T2M2 (10 mg/kg) or anequivalent volume of PBS on study day 1. On study day 4, the mice weresacrificed and blood WBC counts and spleen weights were determined asshown in FIG. 48A. Changes in the percentage of peripheral bloodmononuclear cells (PBMC) CD8⁺ and NKp46⁺ cells were assessed flowcytometry as shown in FIG. 48B. PBMCs were also used to assess NK cellactivity based on lysis of NK-sensitive Yac-1 target cells in a calceinrelease assay as shown in FIG. 48C.

FIG. 49A-FIG. 49B shows results from in vivo assays to determine thedose and temporal responses of various T2 molecules on immune activityin mice. C57BL/6 mice were treated i.v. with equivalent molar IL-15doses of hIL-15 (1 mg/kg), IL15N72D:IL15Rα-Fc (4 mg/kg), T2M2 (variousdoses) or an equivalent volume of PBS on study day 1. On study day 4,the percentage of PBMC CD8⁺ and NKp46⁺ cells were assessed by flowcytometry (FIG. 49A). Nude mice were treated i.v. withIL15N72D/IL15Rα-Fc (0.2 mg/kg) or T2M2 (2 mg/kg) of study day 1. On day4 and 7 post treatment, the percentage of PBMC NKp46⁺ cells was assessedby flow cytometry (FIG. 49B).

FIG. 50A-FIG. 50C shows results from a primary tumor growth model usinga human p53+ HLA-A2+ A375 melanoma cell line in nude mice. A375 humanmelanoma tumor cells (1×10⁶) were injected s.c. into nude mice(5-6/group). Tumors were allowed to establish and mice were treated i.v.with equivalent molar doses of IL-15 (0.35 mg/kg), scTCR-IL15 fusions(1.6 mg/kg), scTCR-IL15/scTCR-IL15Rα complex (3.2 mg/kg), or PBS. Themice were treated three times a week for three weeks starting on studyday 11 (FIG. 50A). A375-tumor bearing nude mice were also treated i.v.with 4 mg/kg T2M as in FIG. 50A (FIG. 50B). A375 tumor bearing nude micewere i.v. with equivalent molar doses of IL-15 (0.2 mg/kg), T2M2 (2mg/kg) or PBS (FIG. 50C). Tumors were measured every other day and tumorvolumes (mean±SEM) were plotted.

FIG. 51A, FIG. 51B, FIG. 51C, and FIG. 51D shows the nucleic acidsequence of c264scTCR/huIL15RαSushi/huIgG1 CH2-CH3 (Fc) construct (alsoreferred to as T2MΔCH1 and T2M2) (SEQ ID NO. 46).

FIG. 52 shows the protein sequence of the maturec264scTCR/huIL15RαSushi/huIgG1 CH2-CH3 (Fc) fusion protein (alsoreferred to as T2MΔCH1 and T2M2) (SEQ ID NO: 47).

FIG. 53A and FIG. 53B shows the nucleic acid sequence of anti-CD20scAb/hIL-15N72D construct (SEQ ID NO: 48).

FIG. 54 shows the protein sequence of the mature anti-CD20scAb/hIL-15N72D fusion protein (SEQ ID NO: 49).

FIG. 55A, FIG. 55B, and FIG. 55C shows the nucleic acid sequence ofanti-CD20 scAb/huIL-15RαSu/huIgG1 Fc construct (SEQ ID NO: 50).

FIG. 56 shows the protein sequence of the mature anti-CD20scAb/huIL-15RαSu/huIgG1 Fc fusion protein (SEQ ID NO: 51).

FIG. 57 shows results from flow cytometry assays to test the CD20antigen specific binding of anti-CD20 scAb T2M molecules to Daudi cells.

FIG. 58 shows results of an assay to determine the antibody dependentcellular cytotoxicity activity mediated by anti-CD20 scAb T2Ms againstCD20⁺ human tumor cells. Various amounts of fusion protein (anti-CD20scAb T2M, c264scTCR T2M (negative control) or chimeric anti-CD20 mAb(positive control)) were mixed with fresh human PBMCs (from 2 differentdonors) and Daudi cells (Calcein labeled) (E:T ratio, 100:1). After anincubation period, the culture medium was collected and analyzedquantitatively for Calcein released from lysed cells.

FIG. 59 shows results of an assay to determine the antibody dependentcellular cytotoxicity activity mediated by anti-CD20 scAb T2Ms againstCD20⁺ human tumor cells. Fusion proteins (anti-CD20 scAb T2M, c264scTCRT2M (negative control) or chimeric anti-CD20 mAb (positive control))were mixed with various rations of fresh human PBMCs (from 2 differentdonors) and Daudi cells (Calcein labeled). After an incubation period,the culture medium was collected and analyzed quantitatively for Calceinreleased from lysed cells.

FIG. 60A and FIG. 60B shows the nucleic acid sequence of anti-CD20 lightchain V domain/human kappa constant domain/hIL-15N72D construct (SEQ IDNO: 52).

FIG. 61 shows the protein sequence of the mature anti-CD20 light chain Vdomain/human kappa constant domain/hIL-15N72D fusion protein (SEQ ID NO:53).

FIG. 62A, FIG. 62B, and FIG. 62C shows the nucleic acid sequence ofanti-CD20 heavy chain V domain/human IgG1 CH1 domain/huIL-15RαSu/huIgG1Fc construct (SEQ ID NO: 54).

FIG. 63 shows the protein sequence of the mature anti-CD20 heavy chain Vdomain/human IgG1 CH1 domain/huIL-15RαSu/huIgG1 Fc fusion protein (SEQID NO: 55).

FIG. 64 is a schematic drawing of IL-15N72D:IL-15RαSu/Fc complex (alsoreferred to as IL-15N72D:IL-15RαSu/huIgG1 CH2-CH3 complex, T2MΔTCRΔCH1and ALT-803). Alt-803 includes IL-15N72D noncovalently associated withthe dimeric IL-15RαSu/Fc fusion protein.

FIG. 65A to FIG. 65D are photographs of gel electrophoresis analysisprofiles of IL-15N72D:IL-15RαSu/Fc (Alt-803) preparations. FIG. 65Ashows IEF pH 3-10 gel analysis. Lane 1, IEF Marker. Lane 2,IL-15N72D:IL-15RαSu/Fc complex (Alt-803) purified by rProtein A column.Lane 3, IL-15RαSu/Fc. Lane 4, IL-15wt. FIG. 65B shows IEF pH3-10 gelanalysis. Lane 1, IEF Marker. Lane 2, IL-15N72D:IL-15RαSu/Fc complex(Alt-803) purified by Q step 1 elution. Lane 3, Q1c by Q step 2 elution.Lane 4, Q2c by Q step 2 elution. FIG. 65C shows SDS-PAGE (reduced)analysis. Lane 1, MW maker. Lane 2, IL-15N72D:IL-15RαSu/Fc complex(Alt-803) purified by rProtein A column. Lane 3, IL-15N72D:IL-15RαSu/Fc(Alt-803) (Q2c) by Q step 2 elution. Lane 4, IL-15RαSu/Fc (from Q flowthrough). FIG. 65D shows SDS-PAGE (reduced) analysis showing proteindeglycosylation. Lane 1, MW markers. Lanes 2 and 3 show N-Glycosidase Fdigested and undigested IL-15N72D:IL-15RαSu/Fc protein, respectively.Lane 4, IL-15 wt.

FIG. 66 is a graph of a SEC chromatogram using Superdex 200 HR 10/30 gelfiltration column. The purified IL-15N72D:IL-15Rα/Fc complex was elutedas a single peak.

FIG. 67 is a graph showing a comparison of the pharmacokinetic profileof IL-15 wt and IL-15N72D:IL-15RαSu/Fc complex following intravenousadministration in CD-1 mice. The anti-IL-15 Ab ELISA measures theconcentration of IL-15 wt (▪). The anti-IL-15 Ab ELISA measures theconcentration of the intact IL-15N72D:IL-15RαSu/Fc molecule (Δ), whereasthe anti-human IgG Fc Ab ELISA measures serum concentration of theIL-15RαSu/Fc fusion protein (▾). The observed concentrations arerepresented by symbols and the model-fitted curves are represented bylines.

FIG. 68 is a graph showing a comparison of the biological activity ofthe in vitro assembled IL-15N72D:IL-15RαSu/Fc (IL-15N72D:IL-15RαSu/FcIVA) with IL-15N72D:IL-15RαSu/Fc. 32Dβ cells were incubated withincreasing concentrations of the in vitro assembledIL-15N72D:IL-15RαSu/Fc (▪) or IL-15N72D:IL-15RαSu/Fc (□) for 72 h priorto addition of WST-1 for 4 h. Cell proliferation was quantitated byabsorbance reading at 440 nm to assess formazan levels. The data pointsshown are means (±standard error) of triplicate samples and the linesrepresent sigmoidal dose-response curve fit for EC₅₀ determination. Theresults are representative of at least three experiments.

FIG. 69 is a set of graphs showing the effect of IL-15 wt andIL-15N72D:IL-15RαSu/Fc (Alt-803) complex on spleen weight and whiteblood cell levels. C57BL/6 mice (5 mice per group) were injectedintravenously with a single dose of IL-15N72D:IL-15RαSu/Fc fusioncomplex (Alt-803) at 1 mg/kg IL-15 wt at 0.28 mg/kg (molar equivalentdose), or PBS as a negative control. Spleen weights (left panel) andwhite blood cell counts in blood (right panel) were determined 4 daysafter injection. The bars represent the mean±standard error (n=5). *P>0.05 compared to PBS and IL-15 wt. The results are representative ofat least two experiments.

FIG. 70 is a set of graphs showing the effect of IL-15 wt andIL-15N72D:IL-15RαSu/Fc complex (Alt-803) on mouse lymphocytes. C57BL/6mice (5 mice per group) were injected intravenously with a single doseof IL-15N72D:IL-15RαSu/Fc fusion complex (Alt-803) at 1 mg/kg, IL-15 wtat 0.28 mg/kg (molar equivalent dose), or PBS as a negative control. Thepercentage of B cells (CD19), CD4 T cells (CD4), NK cells (NKp46) andCD8 T cells (CD8) were determined in splenocytes (left panel:mean±standard error (n=5)) and PBMCs (right panel: levels in pooledblood (n=5)) 4 days after injection. * P>0.05 compared to PBS, ** P>0.05compared to IL-15 wt. The results are representative of at least twoexperiments.

FIG. 71 is a histogram showing the growth pattern of 5T33P murinemyeloma cells in the bone marrow (BM) of C57BL/6NHsd mice. FemaleC57BL/6NHsd mice (n=4-5/group) were injected intravenously (i.v.) with5T33P myeloma cells (1×10⁷) on day 0. Bone marrow (BM) cells werecollected on days 7, 11, 14, 18 and 21 after tumor cell inoculation. BMcells were then stained with phycoerythrin (PE)-conjugated ratanti-mouse IgG2b Ab and evaluated by flow cytometry to determine thepercentage of 5T33P cells in BM. The plotted values represent themean±SE.

FIGS. 72A-FIG. 72D are graphs showing the anti-tumor effects of ALT-803in murine myeloma models. FIG. 72A and FIG. 72B show the effect ofALT-803 or IL-15 on myeloma cells in BM of 5T33P (FIG. 72A) or MOPC-315Pbearing mice. Female mice (5 mice/group) were injected i.v. with 5T33P(FIG. 72A) or MOPC-315P (FIG. 72B) myeloma cells (1×10⁷/mouse) on day 0.ALT-803 (0.2 mg/kg), IL-15 (0.056 mg/kg, an IL-15 molar equivalent doseto 0.2 mg/kg ALT-803), or PBS (dose volume equivalent) was thenadministered as a single i.v. injection on day 15 (5T33P) or 14(MOPC-315P). BM cells were collected 4 days after study drug treatments.The cells were then stained with PE-conjugated rat anti-mouse IgG2b orIgA Ab to evaluate the percentage of 5T33P or MOPC-315P cells in BM,respectively. The plotted values represent the mean±SE; P values arepresented. FIG. 72C shows that ALT-803 treatment prolonged survival ofthe examined mice bearing murine myeloma cells. Female C57BL/6NHsd mice(n=5/group) were injected i.v. with murine 5T33P myeloma cells (1×10⁷cells/mouse) on day 0. A single-dose of ALT-803 was administered i.v. at0.2 mg/kg on day 4. Control mice were treated with PBS on day 4.Survival (or morbidity due to hind leg paralysis) was monitored as astudy endpoint. FIG. 72D shows that ALT-803 treatment prolongs survivalof C57BL/6NHsd mice following subsequent rechallenge with 5T33P myelomacells. 5T33P tumor-bearing mice (n=5) were treated with ALT-803 days 1and 7 or with PBS as in 2A. ALT-803 treated mice that survived (n=4)were rechallenged with 5T33P cells (1×10⁷) on day 93. Fivetreatment-naïve mice were also administered 5T33P cells (1×10⁷) on day93 as a control for tumor development.

FIG. 73A and FIG. 73B are graphs showing ALT-803 activity in murinemyeloma models. FIG. 73A shows effects of ALT-803 on myeloma cells inbone marrow of 5T33P-bearing C57BL/6NHsd mice. Female C57BL/6NHsd mice(n=4/group) were injected i.v. with 5T33P myeloma cells (1×10⁷) on day0. ALT-803 (various dose levels) or PBS was then administered as asingle i.v. injection on day 14. Bone marrow cells were collected 4 daysafter test article treatment (day 18). The cells were then stained withPE-conjugated rat anti-mouse IgG2b Ab and evaluated by flow cytometry todetermine the percentage of 5T33P cells in BM. The plotted valuesrepresent the mean±SE; P values are presented. FIG. 73B shows thatALT-803 treatment prolonged survival of BALB/c mice bearing MOPC-315Pmyeloma cells. Female BALB/c mice (ALT-803-2 doses: n=5; ALT-803-3doses: n=6; PBS: n=6) were injected i.v. with murine MOPC-315P myelomacells (1×10⁷) on day 0. ALT-803 was administered i.v. at 0.2 mg/kg ondays 4 and 11 (ALT-803-2ds group) or on days 4, 7 and 11 (ALT-803-3dsgroup). Control mice were treated with PBS on days 4, 7 and 11. Survival(or morbidity due to hind leg paralysis) was monitored as a studyendpoint.

FIG. 74A and FIG. 74B are histograms showing that 5T33P cell apoptosisis not induced by culturing the cells with ALT-803 or IFNγ. 5T33P cellswere incubated for 24 hours in the presence of media containing (FIG.74A) PBS, ALT-803 (50 and 500 nM) or cisplatin (10 and 100 μM)(Sigma-Aldrich) and (FIG. 74B) PBS, IFN-γ (10, 1, and 0.1 ng/mL) orcisplatin (10 and 100 μM). After staining with FITC Annexin V andpropidium iodine (PI) (BD Bioscience), the cells were analyzed by flowcytometry. Cell viability was assessed as negative staining with FITCAnnexin V and PI. The plotted values represent the mean±SE.

FIG. 75A and FIG. 75B are graphs showing immune cell effects on theanti-myeloma activity of ALT-803. In FIG. 75A, female C57BL/6NHsd mice(n=5/group) were depleted of CD8⁺ T cells (dpCD8), NK1.1⁺ cells (dpNK),or both (dpCD8/NK) by i.p. treatment with anti-CD8 and/or anti-NK1.1 Abson days −2 and −1. Mice were then injected with 5T33P myeloma cells(1×10⁷) on day 0 and treated with anti-CD8 and/or anti-NK1.1 Abs on days7 and 14. The mice were treated with ALT-803 on day 14. Undepleted5T33P-bearing mice receiving PBS served as controls. Four days afterALT-803 treatment, BM cells were isolated and stained withFITC-anti-CD8b, PE-anti-NKp46 and FITC-anti-IgG2b Abs and analyzed byflow cytometry. The percentage of CD8⁺ T cells, NKp46⁺ NK cells andIgG2b⁺ 5T33P myeloma cells in BM are shown. Bars represent the mean±SE.In FIG. 75B, female C57BL/6NHsd mice (n=5/group) were depleted of CD8⁺ Tcells (dpCD8), NK1.1⁺ cells (dpNK), or both (dpCD8/NK) by treatment withanti-CD8 and/or anti-NK1.1 Abs on days −2 and −1 as described in FIG.5A. Mice were then injected with 5T33P myeloma cells (1×10⁷) on day 0and treated with anti-CD8 and/or anti-NK1.1 Abs and then weekly for 8weeks. ALT-803 was administered i.v. at 0.2 mg/kg on days 4 and 11. Micereceiving 5T33P myeloma cells (1×10⁷) on day 0 and PBS on days 4 and 11were used as control. For comparison of ALT-801 vs. PBS or ALT-801+Abdepletion vs. ALT-801, * , P≤0.05; **, P≤0.01; and ***, P≤0.001.

FIG. 76A-FIG. 76D show that ALT-803 induces CD8⁺CD44^(high) memory Tcell proliferation and up-regulation of NKG2D. In FIG. 76A and FIG. 76B,female C57BL/6NHsd mice (5-6 weeks-old, 3 mice/group) were untreated(normal) or injected i.v. with 5T33P myeloma cells (1×10⁷/mouse)(5T33P-bearing) on day 0. ALT-803 (0.2 mg/kg) or PBS (dose volumeequivalent) was administered i.v. on day 14. Four days after treatment,mouse splenocytes were isolated and stained with Abs specific to CD44(PE-Cy7), NKG2D (APC), PD-1 (FITC), CD25 (PE), and CD8 (PerCP-Cy5.5).Stained cells were analyzed by flow cytometry. The percentage ofCD44^(low) and CD44^(high) in CD8⁺ T cells (FIG. 76A) and percentage ofPD-1-, CD25- and NKG2D-positive cells in CD8⁺CD44^(high) memory T cellpopulation (FIG. 76B) are shown. In FIG. 76C, CD3⁺ enriched cells fromspleens of donor C57BL/6NHsd mice were labeled with Celltrace™ Violetand then adoptively transferred (1.5×10⁶ cells/mouse) into syngeneicrecipients (3 mice/group) on day 0 (SD0). On SD2, mice were treated(i.v.) with 0.02 mg/kg of ALT803, 0.2 mg/kg of ALT-803 or PBS (dosevolume equivalent). On SD6, spleens were harvested and analyzedindividually by flow cytometry for donor cells (violet label) andpositive staining with Abs specific to CD44 (PE-Cy7), NKG2D (APC), PD-1(FITC), CD25 (PE), and CD8 (PerCP-Cy5.5). Histograms show proliferationof violet-labeled CD8⁺CD44^(high) memory T cell population. In FIG. 76D,NKG2D^(neg)CD25^(neg)CD8⁺CD44^(high) memory T cells from spleens andlymph nodes of donor C57BL/6NHsd mice were sorted with BD FACS Aria(FIG. 76D) and labeled with Celltrace™ Violet. Donor cells (1×10⁶cells/mouse) were then adoptively transferred into syngeneic recipients(3 mice/group) on SD0. On SD2, mice were treated (i.v.) 0.2 mg/kgALT-803 or PBS (dose volume equivalent). On SD6, spleens were harvestedand analyzed by flow cytometry as described in FIG. 76C. Histograms showproliferation of violet-labeled CD8⁺CD44^(high) memory T cell populationand CD8⁺CD44^(high)NKG2D⁺ and CD8⁺CD44^(high)CD25⁺ subpopulations. Thevalue indicates the percentage of NKG2D⁺ or CD25⁺ cells in the donorCD8⁺CD44^(high) memory T cell population.

FIG. 77A-1, FIG. 77A-2, FIG. 77B-1, and FIG. 77B-2 show a FACS gatingstrategy and analysis of donor NKG2D^(neg)CD25^(neg)CD8⁺CD44^(high) Tcells prior to adoptive transfer (FIG. 77A-1 and FIG. 77A-2) and afteradoptive transfer Celltrace™ Violet labeled cells in ALT-803 treatedmice (FIG. 77B-1 and FIG. 77B-2).

FIG. 78A and FIG. 78B shows that ALT-803 did not increase expressionmaturation markers on BM dendritic cells. Female C57BL/6NHsd mice (5-6weeks-old, 3 mice/group) were untreated (normal) or injected i.v. with5T33P myeloma cells (1×10⁷/mouse) (5T33P-bearing) on day 0. ALT-803 (0.2mg/kg) or PBS (dose volume equivalent) was administered i.v. on day 14.Four days after treatment, BM cells were isolated, pooled and stainedwith Abs specific to CD11c (PE-Cy7), MHC II [I-A(b)] (FITC), CD80(PerCP-Cy5.5), and CD40 (APC), then analyzed by flow cytometry. Micetreated i.p. either with 12.5 μg of LPS (E. coli 055:B5, Sigma-Aldrich)and sacrificed 12 hrs later or with 10 μg of Poly IC (InvivoGene) andsacrificed 24 hrs later served as positive controls. Histograms showstaining with positive Abs (black line) or isotype controls (gray line).

FIG. 79A-FIG. 79D are graphs showing the in vitro cytotoxic activity ofALT-803-treated immune cells. In FIG. 79A, unfractionated or CD8⁺ T cellenriched splenocytes (untouched) from normal C57BL/6NHsd mice (pool of3/group) were cultured with 200 ng/mL of ALT-803 for 72 hrs. Cells werethen harvested, stained with Abs specific to CD44 (PE-Cy7), NKG2D (APC),PD-1 (FITC), CD25 (PE), and CD8 (PerCP-Cy5.5), and analyzed by flowcytometry for expansion of CD8⁺CD44^(high) memory T cell populations. InFIG. 79B and FIG. 79C, unfractionated or CD8⁺ T cell enrichedsplenocytes were activated as described in FIG. 79A, then washedthoroughly and re-plated in duplicate wells (1×10⁶ cells/well)containing 0, 20, or 200 ng/mL ALT-803. NKG2D blocking antibody (10μg/mL) or isotype control antibody (10 μg/mL) was added to appropriatewells as indicated. PHK-67 labeled 5T33P (1×10⁵ cells/well) (FIG. 79B)or A20 tumor cells (1×10⁵ cells/well) (FIG. 79C) were added (E:Tratio=10:1) and incubated for 24 hrs. Target cell killing of theindividual cultures was assessed by analysis of PI staining of PKH-67labeled tumor cells on a BD FACScan. The level of PI staining incultured PHK-67 labeled 5T33P or A20 cells alone served as a backgroundcontrol. In FIG. 79D, in vitro 5T33P killing assay of CD8⁺ T cellenriched spleen cells from normal, IFN-γ KO B6, and perforin KO B6 mice(pool of 3/group). As described above, enriched CD8⁺ T cells (2×10⁷)were incubated with ALT-803 (0.2 μg/mL) for 72 hrs and then re-platedinto triplicated wells (3×10⁶ cells/well) without or with ALT-803 (20ng/ml). PHK-67 labeled 5T33P tumor cells (3×10⁵ cells/well) were addedas target cells (E:T ratio=10:1). After incubation for 20 hrs, targetcell killing was assessed as described above. The percentage ofPI-positive 5T33P cells is shown. Points or bars represent the mean±SE.For comparison of target cells+effector cells vs. target cells alone ortarget cells+KO effector cells vs. target cells+WT effector cells underthe same culture conditions, *, P≤0.05; **, P≤0.01; and ***, P≤0.001.

FIG. 80A-FIG. 80C show that CD8⁺ T cell production of IFN-γ plays a rolein ALT-803-mediated efficacy. In FIG. 80A, ALT-803 induce high level ofserum IFN-γ via CD8⁺ T cells. C57BL/6NHsd mice (n=5) received threedoses of anti-CD8 Ab (dpCD8), anti-NK1.1 Ab (dpNK) or both Abs(dpCD8/NK) i.p. on days −2, −1 and 7. On day 8, a single i.v. dose ofALT-803 (0.2 mg/kg) was administrated and two days later (day 10) serumIFN-γ levels were examined. Bars represent the mean±SE. For comparisonof ALT-803+Ab depletion vs. ALT-803, *, P≤0.05. In FIG. 80B, C57BL/6NHsdmice (n=3) were administrated a single i.v. dose of ALT-803 (0.2 mg/kg)on day 1 or day 2 respectively. On day 3, isolated splenocytes werestained with Abs to CD44 (PE-Cy7), and CD8 (PerCP-Cy5.5), and thenintracellularly stained with FITC-anti-IFN-γ Ab. Dot plots show thepercentage of IFN-γ producing CD8⁺CD44^(high) memory T cells. FIG. 80Cshows that IFN-γ is required for ALT-803 anti-myeloma activity. FemaleIFN-γ KO B6 mice (n=3/group) were injected i.v. with 5T33P myeloma cells(1×10⁷ cells/mouse) on day 0. ALT-803 (0.2 mg/kg) or PBS wasadministered i.v. on days 4 and 11. Survival (or morbidity due to hindleg paralysis) was monitored as a study endpoint.

In FIG. 81A and FIG. 81B, ALT-803 induction of CD8⁺CD44^(high) memory Tcell responses was not dependent on IFN-γ. In FIG. 81A and FIG. 81B,enriched CD8⁺ T cells (positive selection) from splenocytes and lymphnodes of IFN-γ KO B6 mice (6 weeks old) were labeled with Celltrace™Violet and adoptively transferred (1.5×10⁶ cell/mouse) into IFN-γ KO B6recipients (KO, n=5) (FIG. 81A) or wild-type C57BL/6NHsd recipients (WT,n=5) (FIG. 81B) on day 0 (SD0). On SD2, 3 KO and 3 WT mice were treatedwith 0.2 mg/kg ALT-803 (i.v.) and the remaining 2 KO and 2 WT micereceived PBS (i.v.) as controls. On SD6, spleens were harvested andanalyzed individually by flow cytometry for donor cells (violet label)and positive staining with Abs specific to CD44 (PE-Cy7), NKG2D (APC),and CD8 (PerCP-Cy5.5). Histograms show proliferation of violet-labeledCD8⁺CD44^(high) and CD8⁺CD44^(high)NKG2D⁺ memory T cell population.

FIG. 82 shows a tumor growth curve. C57BL/6 mice (8-10 weeks old) (5mice/group) were injected subcutaneously (s.c.) with EG7-OVA cells(1×106 cells/mouse) on study day 0. ALT-803 (0.415, or 0.83 mg/kg),rhlL-15 (0.06 mg/kg) or PBS was administered i.v. on 1, 4, 8, and 11days post tumor cell injection. Tumor volumes were measured and themean±SEM were plotted. Treatment with ALT-803 at the two dosing levelsas well as rhlL-15 significantly inhibited EG7-OVA tumor growth. Two-wayANOVA data analyses are shown in the table beneath the graph.

FIG. 83 shows a graphical presentation of tumor growth inhibition ofALT-803 versus PBS (top panel) or versus rhlL-15 (bottom panel)treatment. ALT-803 treatment at 0.415 and 0.83 mg/kg resulted in 63.5%and 68.3% TGI over PBS, and 47.1% and 54.1% TGI over rhlL-15. treatment13

FIG. 84 is a graph showing that ALT-803 treatment did not cause mousebody weight reduction. EG7-OVA tumor bearing mice were treated withALT-803 at 0.415 mg/kg or 0.83 mg/kg, or rhlL-15 at 0.06 mg/kg, alongwith PBS treatment as a control for 4 iv injections on 1, 4, 8, and 11days post tumor cell injection.

FIG. 85 shows that Alt-803 significantly inhibited HIV infection in anin vivo mouse model.

FIG. 86A, FIG. 86B, and FIG. 86C provides the amino acid sequence of theproteins making up Alt-803, as well as the nucleic acid sequence of thepolynucleotide encoding Alt-803. Alt-803 is referred to asIL-15N72D:IL-15RαSu/Fc complex, huIL15N72D:huIL15RαSushi/huIgG1 CH2-CH3,IL-15N72D:IL-15Rα-IgG CH2-CH3, T2MΔTCRΔCH1 and ALT-803 at various pointsin the application. FIG. 86A, FIG. 86B, and FIG. 86C provide SEQ ID NOs:56, 3, 57 and 4, respectively.

FIG. 87 depicts an anti-CD20 scFv/IL-15:anti-CD20 scFv/ IL-15Rα/IgG Fcprotein complex (2B8T2M) which comprises scFv anti-CD20 Ab domainslinked to IL-15 and IL-15Rα/Fc domains. This complex mediates anti-Bcell lymphoma activity through Fc-dependent ADCC and CDC andFc-independent direct cell killing, and further enhances effectorresponses by IL-15 activation of IL-15Rβγc-bearing immune cells

FIG. 88A and FIG. 88B show molecular weight analysis of the anti-CD20scFv/IL-15:anti-CD20 scFv/ IL-15Rα/IgG Fc protein complex (2B8T2M). FIG.88A shows reduced SDS-PAGE analysis of purified rituximab (C2B8, lane 1)and anti-CD20 scAb/IL-15:anti-CD20 scAb/IL-15Rα/IgG Fc protein complex(2B8T2M, lane 2). FIG. 88B shows size exclusion chromatography (SEC)analysis of purified rituximab (C2B8, top panel) and anti-CD20scAb/IL-15:anti-CD20 scAb/IL-15Rα/IgG Fc protein complex (2B8T2M, bottompanel).

FIG. 89A and FIG. 89B show functional activity of IL-15 and anti-CD20scFv domains of anti-CD20 scFv/IL-15:anti-CD20 scFv/ IL-15Rα/IgG Fcprotein complexes. FIG. 89A is a graph showing effects of 2B8T2M proteincomplexes on proliferation of IL-15 dependent 32Dβ cells compared to264T2M and 268T2M fusion proteins. FIG. 89B shows results from flowcytometry assays to test the CD20 antigen specific binding of anti-CD20scFv/IL-15:anti-CD20 scFv/ IL-15Rα/IgG Fc protein complexes (2B8T2M) toDaudi cells. Staining of CD20+ human Daudi lymphoma cells was performedwith 50 nM fusion proteins.

FIG. 90 is a graph depicting antibody-dependent cellular cytotoxicity(ADCC) activity of 2B8T2M complex. Purified fusion proteins were mixedwith purified human T cells+NK cells and incubated 2 hrs with calceinlabeled Daudi cells at a 20:1 E:T ratio. Cell lysis was determined bycalcein release (Mosquera et al., J Immunol, 174: 4381-4388, 2005).

FIG. 91 is a graph depicting complement dependent cytotoxicity (CDC)activity of 2B8T2M complex. Purified proteins (concentrations, asindicated) were mixed with human serum and incubated 2 hrs with Daudicells. Cell death was assessed by flow cytometry following staining withFITC-Annexin-V and propidium iodide (PI).

FIG. 92 is a graph depicting programed cell death (PCD) activity of2B8T2M complex. Purified proteins (10 nM) were incubated 2 days withDaudi cells. Cell death was assessed by flow cytometry as described inFIG. 5.

FIG. 93 is a graph depicting anti-lymphoma activity of 2B8T2M complex.Purified proteins (concentrations, as indicated) were mixed withpurified human T cells+NK cells, and incubated 2 days withPKH67-labelled Daudi cells at a low 2:1 E:T ratio. Daudi cell death wasdetermined by flow cytometry following staining with propidium iodide(PI).

FIG. 94 depicts the detection of Daudi cells in bone marrow oftumor-bearing SCID mice. Female SCID mice (C.B-17/IcrHsd-Prkdc-scid)were injected i.v. with 107 Daudi cells or HBSS (control). After 2weeks, animals were sacrificed and femoral bone marrow cells werecollected. The cells were stained with PE-conjugated anti-HLA-DR mAb todetect Daudi cells in tumor-bearing mice.

FIG. 95 depicts an efficacy study of 2B8T2M against Daudi B Lymphoma inSCID Mice.

FIG. 96 is a graph showing that anti-Daudi Activity of 2B8T2M is morepotent than C2B8 in SCID Mice.

FIG. 97 is a graph showing immunostimulatory spleen enlargement inducedby 2B8T2M in SCID Mice.

FIG. 98 is a graph showing induction of NK Cells by 2B8T2M in SCID Mice.

FIG. 99 is a graph showing extended half-life of 2B8T2M as determined byconcentration in blood on day-4 after treatment.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions featuring ALT-803, a complex of aninterleukin-15 (IL-15) superagonist mutant and a dimeric IL-15 receptorα/Fc fusion protein, and methods of using such compositions to enhancean immune response against a neoplasia (e.g., multiple myeloma,beta-cell lymphoma, urothelial/bladder carcinoma and melanoma) or aviral infection (e.g., human immunodeficiency virus).

The invention is based, at least in part, on the discovery that ALT-803exhibited significantly stronger in vivo biological activity on NK and Tcells than IL-15. As reported in more detail below, a single dose ofALT-803, but not IL-15 alone, eliminated well-established 5T33P andMOPC-315P myeloma cells in the bone marrow of tumor-bearing mice.Treatment with ALT-803 also significantly prolonged survival ofmyeloma-bearing mice and provided resistance to rechallenge with thesame tumor cells through a CD8⁺ T cell-dependent mechanism. ALT-803treatment stimulated CD8⁺ T cells to secrete large amounts ofinterferon-γ (IFN-γ) and promoted rapid expansion of CD8⁺CD44^(high)memory T cells in vivo. These memory CD8⁺ T cells exhibitedALT-803-mediated up-regulation of NKG2D but not PD-1 or CD25 on theircell surfaces. ALT-803-activated CD8⁺ memory T cells also exhibitednon-specific in-vitro cytotoxicity against myeloma and other tumorcells, whereas IFN-γ had no direct effect on myeloma cell growth.ALT-803 lost its anti-myeloma activity in tumor-bearing IFN-γ-knockoutmice, but retained the ability to promote the proliferation ofCD8⁺CD44^(high) memory T cells, indicating that the stimulation ofCD8⁺CD44^(high) memory T cells by ALT-803 is IFN-γ-independent. Thus,besides well-known IL-15 biological functions in host immunity, theresults reported in detail below demonstrate that IL-15-based ALT-803could activate CD8⁺CD44^(high) memory T cells to acquire a uniqueinnate-like phenotype and secrete IFN-γ for non-specific tumor-cellkilling. This unique immune modulatory property of ALT-803 provides forits use as a promising novel immunotherapeutic agent against cancer andviral infections.

The invention is also based, at least in part, on the discovery thatAlt-803 inhibited lymphoma tumor growth in an in vivo mouse model oflymphoma.

The invention is also based, at least in part, on the discovery thatAlt-803 inhibited HIV infection in an in vivo mouse model.

In other embodiments, the invention provides compositions comprising2B8T2M for the treatment of lymphomas.

Alt-803

Alt-803 comprises a novel IL-15 mutant with increased ability to bindIL-2Rβγ and enhanced biological activity. This super agonist mutant ofIL-15 was described in a publication (J Immunol 2009 183:3598) and apatent has been issued by the U.S. Patent & Trademark Office on thesuper agonist and several patents applications are pending (e.g., U.S.Ser. Nos. 12/151,980 and 13/238,925). This IL-15 super agonist incombination with a soluble IL-15α receptor fusion protein (IL-15Rα-Fc)results in a protein complex with highly potent IL-15 activity in vitroand in vivo. This IL-15 super agonist complex (IL-15N72D/IL-15Rα-Fc) isreferred to as ALT-803. Pharmacokinetic analysis indicated that thecomplex has a half-life in mice of 25 hours following i.v.administration. As reported in detail herein below, ALT-803 exhibitsimpressive anti-tumor activity against aggressive solid andhematological tumor models in immunocompetent mice. It can beadministered as a monotherapy using a weekly i.v. dose regimen. TheALT-803 anti-tumor response is also durable. Tumor-bearing mice thatwere cured after ALT-803 treatment were also highly resistant tore-challenge with the same tumor cells indicating that ALT-803 induceseffective immunological memory responses against the re-introduced tumorcells.

Interleukin-15

Interleukin-15 (IL-15) is an important cytokine for the development,proliferation and activation of effector NK cells and CD8⁺ memory Tcells. IL-15 binds to the IL-15 receptor α (IL-15Rα) and is presented intrans to the IL-2/IL-15 receptor β-common γ chain (IL-15Rβγ_(c)) complexon effector cells. IL-15 and IL-2 share binding to the IL-15Rβγ_(c) andsignal through STAT3 and STAT5 pathways. However, unlike IL-2, IL-15does not support maintenance of CD4⁺CD25⁺FoxP3⁺ regulatory T (Treg)cells or induce cell death of activated CD8⁺ T cells, effects that mayhave limited the therapeutic activity of IL-2 against multiple myeloma.Additionally, IL-15 is the only cytokine known to provide anti-apoptoticsignaling to effector CD8⁺ T cells. IL-15, either administered alone oras a complex with the IL-15Rα, exhibits potent anti-tumor activitiesagainst well-established solid tumors in experimental animal models and,thus, has been identified as one of the most promising immunotherapeuticdrugs that could potentially cure cancer. However, there have been noreports showing efficacy of IL-15 against hematologic tumors.

To facilitate clinical development of an IL-15-based cancer therapeutic,a novel IL-15 mutant with increased biological activity compared toIL-15 was identified (Zhu et al., J Immunol, 183: 3598-3607, 2009). Thepharmacokinetics and biological activity of this IL-15 super-agonist(IL-15N72D) was further improved by the creation of IL-15N72D:IL-15Rα/Fcfusion complex (ALT-803), such that the super agonist complex has atleast 25-times the activity of the native cytokine in vivo (Han et al.,Cytokine, 56: 804-810, 2011). The results reported herein below alsorevealed that ALT-803 employs a novel mechanism of action againstmyeloma.

Fc Domain

Alt-803 comprises an IL-15N72D:IL-15Rα/Fc fusion complex. Fusionproteins that combine the Fc regions of IgG with the domains of anotherprotein, such as various cytokines and soluble receptors have beenreported (see, for example, Capon et al., Nature, 337:525-531, 1989;Chamow et al., Trends Biotechnol., 14:52-60, 1996); U.S. Pat. Nos.5,116,964 and 5,541,087). The prototype fusion protein is a homodimericprotein linked through cysteine residues in the hinge region of IgG Fc,resulting in a molecule similar to an IgG molecule without the heavychain variable and C_(H)1 domains and light chains. The dimeric natureof fusion proteins comprising the Fc domain may be advantageous inproviding higher order interactions (i.e. bivalent or bispecificbinding) with other molecules. Due to the structural homology, Fc fusionproteins exhibit an in vivo pharmacokinetic profile comparable to thatof human IgG with a similar isotype. Immunoglobulins of the IgG classare among the most abundant proteins in human blood, and theircirculation half-lives can reach as long as 21 days. To extend thecirculating half-life of IL-15 or an IL-15 fusion protein and/or toincrease its biological activity, fusion protein complexes containingthe IL-15 domain non-covalently bound to IL-15Rα covalently linked tothe Fc portion of the human heavy chain IgG protein have been made(e.g., Alt-803).

The term “Fc” refers to a non-antigen-binding fragment of an antibody.Such an “Fc” can be in monomeric or multimeric form. The originalimmunoglobulin source of the native Fc is preferably of human origin andmay be any of the immunoglobulins, although IgG1 and IgG2 are preferred.Native Fc's are made up of monomeric polypeptides that may be linkedinto dimeric or multimeric forms by covalent (i.e., disulfide bonds) andnon-covalent association. The number of intermolecular disulfide bondsbetween monomeric subunits of native Fc molecules ranges from 1 to 4depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2,IgG3, IgAl, IgGA2). One example of a native Fc is a disulfide-bondeddimer resulting from papain digestion of an IgG (see Ellison et al.(1982), Nucleic Acids Res. 10: 4071-9). The term “native Fc” as usedherein is generic to the monomeric, dimeric, and multimeric forms. Fcdomains containing binding sites for Protein A, Protein G, various Fcreceptors and complement proteins

In some embodiments, the term “Fc variant” refers to a molecule orsequence that is modified from a native Fc, but still comprises abinding site for the salvage receptor, FcRn.

International applications WO 97/34631 (published Sep. 25, 1997) and WO96/32478 describe exemplary Fc variants, as well as interaction with thesalvage receptor, and are hereby incorporated by reference. Thus, theterm “Fc variant” comprises a molecule or sequence that is humanizedfrom a non-human native Fc. Furthermore, a native Fc comprises sitesthat may be removed because they provide structural features orbiological activity that are not required for the fusion molecules ofthe present invention. Thus, in certain embodiments, the term “Fcvariant” comprises a molecule or sequence that lacks one or more nativeFc sites or residues that affect or are involved in (1) disulfide bondformation, (2) incompatibility with a selected host cell (3) N-terminalheterogeneity upon expression in a selected host cell, (4)glycosylation, (5) interaction with complement, (6) binding to an Fcreceptor other than a salvage receptor, or (7) antibody-dependentcellular cytotoxicity (ADCC). Fc variants are described in furtherdetail hereinafter.

The term “Fc domain” encompasses native Fc and Fc variant molecules andsequences as defined above. As with Fc variants and native Fc's, theterm “Fc domain” includes molecules in monomeric or multimeric form,whether digested from whole antibody or produced by recombinant geneexpression or by other means.

Fusions Protein Complexes

The invention provides Alt-803, which is a fusion protein complex, and2B8T2M. In certain embodiments, the first fusion protein comprises afirst biologically active polypeptide covalently linked tointerleukin-15 (IL-15) or functional fragment thereof; and the secondfusion protein comprises a second biologically active polypeptidecovalently linked to soluble interleukin-15 receptor alpha (IL-15Rα)polypeptide or functional fragment thereof, where the IL-15 domain of afirst fusion protein binds to the soluble IL-15Rα domain of the secondfusion protein to form a soluble fusion protein complex. Fusion proteincomplexes of the invention also comprise immunoglobulin Fc domain or afunctional fragment thereof linked to one or both of the first andsecond fusion proteins. Preferably the Fc domains linked to the firstand second fusion proteins interact to form a fusion protein complex.Such a complex may be stabilized by disulfide bond formation between theimmunoglobulin Fc domains. In certain embodiments, the soluble fusionprotein complexes of the invention include an IL-15 polypeptide, IL-15variant or a functional fragment thereof and a soluble IL-15Rαpolypeptide or a functional fragment thereof, wherein one or both of theIL-15 and IL-15Rα polypeptides further include an immunoglobulin Fcdomain or a functional fragment thereof.

In certain examples, one of the biologically active polypeptidescomprises a first soluble TCR or fragment thereof. The other or secondbiologically active polypeptide comprises the first soluble TCR orfunctional fragment thereof and thus creates a multivalent TCR fusionprotein complex with increased binding activity for cognate ligandscompared to the monovalent TCR. Further, the other biologically activepolypeptide comprises a second soluble TCR or functional fragmentthereof, different than the first soluble TCR. In certain examples, TCRsare produced that have higher affinity, or increased binding affinityfor cognate ligands as compared, for example, to the native TCR. If thesoluble TCR of the invention as described herein has a higher avidity oraffinity for its ligand, then it is useful as a specific probe forcell-surface bound antigen. In certain preferred examples of theinvention, the TCR is specific for recognition of a particular antigen.

In exemplary embodiments, TCR is a heterodimer comprising an α chain(herein referred to as a, alpha, or a chain) and a β chain (hereinreferred to as β, beta, or b chain). In other exemplary embodiments, theTCR comprises a single chain TCR polypeptide. The single chain TCR maycomprise a TCR V-α chain covalently linked to a TCR V-β chain by apeptide linker sequence. The single chain TCR may further comprise asoluble TCR Cβ chain fragment covalently linked to a TCR V-β chain. Thesingle chain TCR may further comprise a soluble TCR Cα chain fragmentcovalently linked to a TCR V-α chain.

In a further embodiment, one or both of the first and secondbiologically active polypeptides comprises an antibody or functionalfragment thereof.

In another embodiment, the antigen for the TCR domain comprises peptideantigen presented in an MHC or HLA molecule. In a further embodiment,the peptide antigen is derived from a tumor associated polypeptide orvirus encoded polypeptide.

In another embodiment, the antigen for the antibody domain comprises acell surface receptor or ligand.

In a further embodiment, the antigen comprises a CD antigen, cytokine orchemokine receptor or ligand, growth factor receptor or ligand, tissuefactor, cell adhesion molecule, MHC/MHC-like molecules, Fc receptor,Toll-like receptor, NK receptor, TCR, BCR, positive/negativeco-stimulatory receptor or ligand, death receptor or ligand, tumorassociated antigen, or virus encoded antigen.

As used herein, the term “biologically active polypeptide” or “effectormolecule” is meant an amino acid sequence such as a protein, polypeptideor peptide; a sugar or polysaccharide; a lipid or a glycolipid,glycoprotein, or lipoprotein that can produce the desired effects asdiscussed herein. Effector molecules also include chemical agents. Alsocontemplated are effector molecule nucleic acids encoding a biologicallyactive or effector protein, polypeptide, or peptide. Thus, suitablemolecules include regulatory factors, enzymes, antibodies, or drugs aswell as DNA, RNA, and oligonucleotides. The biologically activepolypeptides or effector molecule can be naturally-occurring or it canbe synthesized from known components, e.g., by recombinant or chemicalsynthesis and can include heterologous components. A biologically activepolypeptides or effector molecule is generally between about 0.1 to 100KD or greater up to about 1000 KD, preferably between about 0.1, 0.2,0.5, 1, 2, 5, 10, 20, 30 and 50 KD as judged by standard molecule sizingtechniques such as centrifugation or SDS-polyacrylamide gelelectrophoresis. Desired effects of the invention include, but are notlimited to, for example, forming a fusion protein complex of theinvention with increased binding activity, killing a target cell, e.g.either to induce cell proliferation or cell death, initiate an immuneresponse, in preventing or treating a disease, or to act as a detectionmolecule for diagnostic purposes. For such detection, an assay could beused, for example an assay that includes sequential steps of culturingcells to proliferate same, and contacting the cells with a TCR fusioncomplex of the invention and then evaluating whether the TCR fusioncomplex inhibits further development of the cells.

Covalently linking the effector molecule to the fusion protein complexesof the invention in accordance with the invention provides a number ofsignificant advantages. Fusion protein complexes of the invention can beproduced that contain a single effector molecule, including such apeptide of known structure. Additionally, a wide variety of effectormolecules can be produced in similar DNA vectors. That is, a library ofdifferent effector molecules can be linked to the fusion proteincomplexes for recognition of infected or diseased cells. Further, fortherapeutic applications, rather than administration of a the fusionprotein complex of the invention to a subject, a DNA expression vectorcoding for the fusion protein complex can be administered for in vivoexpression of the fusion protein complex. Such an approach avoids costlypurification steps typically associated with preparation of recombinantproteins and avoids the complexities of antigen uptake and processingassociated with conventional approaches.

As noted, components of the fusion proteins disclosed herein, e.g.,effector molecule such as cytokines, chemokines, growth factors, proteintoxins, immunoglobulin domains or other bioactive molecules and anypeptide linkers, can be organized in nearly any fashion provided thatthe fusion protein has the function for which it was intended. Inparticular, each component of the fusion protein can be spaced fromanother component by at least one suitable peptide linker sequence ifdesired. Additionally, the fusion proteins may include tags, e.g., tofacilitate modification, identification and/or purification of thefusion protein. More specific fusion proteins are in the Examplesdescribed below.

Pharmaceutical Therapeutics

The invention provides pharmaceutical compositions comprising Alt-803 or2B8T2M for use as a therapeutic. For therapeutic uses, Alt-803 or 2B8T2Mmay be administered systemically, for example, formulated in apharmaceutically-acceptable buffer such as physiological saline.Preferable routes of administration include, for example, instillationinto the bladder, subcutaneous, intravenous, interperitoneally,intramuscular, or intradermal injections that provide continuous,sustained levels of the drug in the patient. Treatment of human patientsor other animals will be carried out using a therapeutically effectiveamount of a therapeutic identified herein in aphysiologically-acceptable carrier. Suitable carriers and theirformulation are described, for example, in Remington's PharmaceuticalSciences by E. W. Martin. The amount of the therapeutic agent to beadministered varies depending upon the manner of administration, the ageand body weight of the patient, and with the clinical symptoms of theneoplasia. Generally, amounts will be in the range of those used forother agents used in the treatment of other diseases associated withneoplasia, although in certain instances lower amounts will be neededbecause of the increased specificity of the compound. A compound isadministered at a dosage that enhances an immune response of a subject,or that reduces the proliferation, survival, or invasiveness of aneoplastic cell as determined by a method known to one skilled in theart. In other embodiments, the compound is administered at a dosage thatreduces infection by a virus of interest.

Formulation of Pharmaceutical Compositions

The administration of Alt-803 or 2B8T2M for the treatment of a neoplasiamay be by any suitable means that results in a concentration of thetherapeutic that, combined with other components, is effective inameliorating, reducing, or stabilizing a neoplasia. Alt-803 or 2B8T2Mmay be contained in any appropriate amount in any suitable carriersubstance, and is generally present in an amount of 1-95% by weight ofthe total weight of the composition. The composition may be provided ina dosage form that is suitable for parenteral (e.g., subcutaneously,intravenously, intramuscularly, intravesicularly or intraperitoneally)administration route. The pharmaceutical compositions may be formulatedaccording to conventional pharmaceutical practice (see, e.g., Remington:The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro,Lippincott Williams & Wilkins, 2000 and Encyclopedia of PharmaceuticalTechnology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, MarcelDekker, New York).

Human dosage amounts can initially be determined by extrapolating fromthe amount of compound used in mice, as a skilled artisan recognizes itis routine in the art to modify the dosage for humans compared to animalmodels. In certain embodiments it is envisioned that the dosage may varyfrom between about 1 μg compound/Kg body weight to about 5000 mgcompound/Kg body weight; or from about 5 mg/Kg body weight to about 4000mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kgbody weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg bodyweight; or from about 100 mg/Kg body weight to about 1000 mg/Kg bodyweight; or from about 150 mg/Kg body weight to about 500 mg/Kg bodyweight. In other embodiments this dose may be about 1, 5, 10, 25, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000,4500, or 5000 mg/Kg body weight. In other embodiments, it is envisagedthat doses may be in the range of about 5 mg compound/Kg body to about20 mg compound/Kg body. In other embodiments the doses may be about 8,10, 12, 14, 16 or 18 mg/Kg body weight. Of course, this dosage amountmay be adjusted upward or downward, as is routinely done in suchtreatment protocols, depending on the results of the initial clinicaltrials and the needs of a particular patient.

In particular embodiments, Alt-803 and 2B8T2M are formulated in anexcipient suitable for parenteral administration. In particularembodiments, 2B8T2M is administered at 0.5 mg/kg-about 10 mg/kg (e.g.,0.5, 1, 3, 5, 10 mg/kg).

For the treatment of bladder cancer, Alt-803 is administered byinstillation into the bladder. Methods of instillation are known. See,for example, Lawrencia, et al., Gene Ther 8, 760-8 (2001); Nogawa, etal., J Clin Invest 115, 978-85 (2005); Ng, et al., Methods Enzymol 391,304-13 2005; Tyagi, et al., J Urol 171, 483-9 (2004); Trevisani, et al.,J Pharmacol Exp Ther 309, 1167-73 (2004); Trevisani, et al., NatNeurosci 5, 546-51 (2002)); (Segal, et al., 1975). (Dyson, et al.,2005). (Batista, et al., 2005; Dyson, et al., 2005).

Pharmaceutical compositions are formulated with appropriate excipientsinto a pharmaceutical composition that, upon administration, releasesthe therapeutic in a controlled manner. Examples include single ormultiple unit tablet or capsule compositions, oil solutions,suspensions, emulsions, microcapsules, microspheres, molecularcomplexes, nanoparticles, patches, and liposomes.

Parenteral Compositions

The pharmaceutical composition comprising Alt-803 or 2B8T2M may beadministered parenterally by injection, infusion or implantation(subcutaneous, intravenous, intramuscular, intravesicularly,intraperitoneal, or the like) in dosage forms, formulations, or viasuitable delivery devices or implants containing conventional, non-toxicpharmaceutically acceptable carriers and adjuvants. The formulation andpreparation of such compositions are well known to those skilled in theart of pharmaceutical formulation. Formulations can be found inRemington: The Science and Practice of Pharmacy, supra.

Compositions comprising Alt-803 or 2B8T2M for parenteral use may beprovided in unit dosage forms (e.g., in single-dose ampoules), or invials containing several doses and in which a suitable preservative maybe added (see below). The composition may be in the form of a solution,a suspension, an emulsion, an infusion device, or a delivery device forimplantation, or it may be presented as a dry powder to be reconstitutedwith water or another suitable vehicle before use. Apart from the activeagent that reduces or ameliorates a neoplasia, the composition mayinclude suitable parenterally acceptable carriers and/or excipients. Theactive therapeutic agent(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease. Furthermore, the composition may include suspending,solubilizing, stabilizing, pH-adjusting agents, tonicity adjustingagents, and/or dispersing, agents.

As indicated above, the pharmaceutical compositions comprising Alt-803or 2B8T2M may be in a form suitable for sterile injection. To preparesuch a composition, the suitable active antineoplastic therapeutic(s)are dissolved or suspended in a parenterally acceptable liquid vehicle.Among acceptable vehicles and solvents that may be employed are water,water adjusted to a suitable pH by addition of an appropriate amount ofhydrochloric acid, sodium hydroxide or a suitable buffer,1,3-butanediol, Ringer's solution, and isotonic sodium chloride solutionand dextrose solution. The aqueous formulation may also contain one ormore preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate).In cases where one of the compounds is only sparingly or slightlysoluble in water, a dissolution enhancing or solubilizing agent can beadded, or the solvent may include 10-60% w/w of propylene glycol or thelike.

The present invention provides methods of treating neoplastic diseaseand/or disorders or symptoms thereof which comprise administering atherapeutically effective amount of a pharmaceutical compositioncomprising a compound of the formulae herein to a subject (e.g., amammal such as a human). Thus, one embodiment is a method of treating asubject suffering from or susceptible to a neoplastic disease ordisorder or symptom thereof. The method includes the step ofadministering to the mammal a therapeutic amount of an amount of acompound herein sufficient to treat the disease or disorder or symptomthereof, under conditions such that the disease or disorder is treated.

The methods herein include administering to the subject (including asubject identified as in need of such treatment) an effective amount ofa compound described herein, or a composition described herein toproduce such effect. Identifying a subject in need of such treatment canbe in the judgment of a subject or a health care professional and can besubjective (e.g. opinion) or objective (e.g. measurable by a test ordiagnostic method).

The therapeutic methods of the invention (which include prophylactictreatment) in general comprise administration of a therapeuticallyeffective amount of the compounds herein, such as a compound of theformulae herein to a subject (e.g., animal, human) in need thereof,including a mammal, particularly a human. Such treatment will besuitably administered to subjects, particularly humans, suffering from,having, susceptible to, or at risk for a neoplastic disease, disorder,or symptom thereof. Determination of those subjects “at risk” can bemade by any objective or subjective determination by a diagnostic testor opinion of a subject or health care provider (e.g., genetic test,enzyme or protein marker, Marker (as defined herein), family history,and the like). Alt-803 or 2B8T2M may be used in the treatment of anyother disorders in which an increase in an immune response is desired.

In one embodiment, the invention provides a method of monitoringtreatment progress. The method includes the step of determining a levelof diagnostic marker (Marker) (e.g., any target delineated hereinmodulated by a compound herein, a protein or indicator thereof, etc.) ordiagnostic measurement (e.g., screen, assay) in a subject suffering fromor susceptible to a disorder or symptoms thereof associated withneoplasia in which the subject has been administered a therapeuticamount of a compound herein sufficient to treat the disease or symptomsthereof. The level of Marker determined in the method can be compared toknown levels of Marker in either healthy normal controls or in otherafflicted patients to establish the subject's disease status. Inpreferred embodiments, a second level of Marker in the subject isdetermined at a time point later than the determination of the firstlevel, and the two levels are compared to monitor the course of diseaseor the efficacy of the therapy. In certain preferred embodiments, apre-treatment level of Marker in the subject is determined prior tobeginning treatment according to this invention; this pre-treatmentlevel of Marker can then be compared to the level of Marker in thesubject after the treatment commences, to determine the efficacy of thetreatment.

Combination Therapies

Optionally, an anti-neoplasia therapeutic, such as Alt-803 or 2B8T2M,may be administered in combination with any other standardanti-neoplasia therapy or conventional chemotherapeutic agent, such asan alkylating agent; such methods are known to the skilled artisan anddescribed in Remington's Pharmaceutical Sciences by E. W. Martin. Ifdesired, Alt-803 is administered in combination with any conventionalanti-neoplastic therapy, including but not limited to, surgery,radiation therapy, or chemotherapy.

Kits or Pharmaceutical Systems

Pharmaceutical compositions comprising Alt-803 or 2B8T2M may beassembled into kits or pharmaceutical systems for use in ameliorating aneoplasia. Kits or pharmaceutical systems according to this aspect ofthe invention comprise a carrier means, such as a box, carton, tube orthe like, having in close confinement therein one or more containermeans, such as vials, tubes, ampoules, bottles and the like. The kits orpharmaceutical systems of the invention may also comprise associatedinstructions for using Alt-803 or 2B8T2M.

Linkers

The fusion complexes of the invention preferably also include a flexiblelinker sequence interposed between the IL-15 or IL-15Rα domains and thebiologically active polypeptide. The linker sequence should alloweffective positioning of the biologically active polypeptide withrespect to the IL-15 or IL-15Rα domains to allow functional activity ofboth domains. In embodiments where the biologically active polypeptideis a TCR, the linker sequence positions the TCR molecule binding grooveso that the T cell receptor can recognize presenting MHC-peptidecomplexes and can deliver the effector molecule to a desired site.Successful presentation of the effector molecule can modulate theactivity of a cell either to induce or to inhibit T-cell proliferation,or to initiate or inhibit an immune response to a particular site, asdetermined by the assays disclosed below, including the in vitro assaysthat includes sequential steps of culturing T cells to proliferate same,and contacting the T cells with a TCR fusion complex of the inventionand then evaluating whether the TCR fusion complex inhibits furtherdevelopment of the cells.

In certain embodiments, the soluble fusion protein complex has a linkerwherein the first biologically active polypeptide is covalently linkedto IL-15 (or functional fragment thereof) by polypeptide linkersequence.

In other certain embodiments, the soluble fusion protein complex asdescribed herein has a linker wherein the second biologically activepolypeptide is covalently linked to IL-15Rα polypeptide (or functionalfragment thereof) by polypeptide linker sequence.

The linker sequence is preferably encoded by a nucleotide sequenceresulting in a peptide that can effectively position the binding grooveof a TCR molecule for recognition of a presenting antigen or the bindingdomain of an antibody molecule for recognition of an antigen. As usedherein, the phrase “effective positioning of the biologically activepolypeptide with respect to the IL-15 or IL-15Rα domains”, or othersimilar phrase, is intended to mean the biologically active polypeptidelinked to the IL-15 or IL-15Rα domains is positioned so that the IL-15or IL-15Rα domains are capable of interacting with each other to form aprotein complex. In certain embodiments, the IL-15 or IL-15Rα domainsare effectively positioned to allow interactions with immune cells toinitiate or inhibit an immune reaction, or to inhibit or stimulate celldevelopment.

The fusion complexes of the invention preferably also include a flexiblelinker sequence interposed between the IL-15 or IL-15Rα domains and theimmunoglobulin Fc domain. The linker sequence should allow effectivepositioning of the Fc domain, biologically active polypeptide and IL-15or IL-15Rα domains to allow functional activity of each domain. Incertain embodiments, the Fc domains are effectively positioned to allowproper fusion protein complex formation and/or interactions with Fcreceptors on immune cells or proteins of the complement system tostimulate Fc-mediated effects including opsonization, cell lysis,degranulation of mast cells, basophils, and eosinophils, and other Fcreceptor-dependent processes; activation of the complement pathway; andenhanced in vivo half-life of the fusion protein complex.

Linker sequences can also be used to link two or more polypeptides ofthe biologically active polypeptide to generated a single-chain moleculewith the desired functional activity.

Preferably the linker sequence comprises from about 7 to 20 amino acids,more preferably from about 8 to 16 amino acids. The linker sequence ispreferably flexible so as not hold the biologically active polypeptideor effector molecule in a single undesired conformation. The linkersequence can be used, e.g., to space the recognition site from the fusedmolecule. Specifically, the peptide linker sequence can be positionedbetween the biologically active polypeptide and the effector molecule,e.g., to chemically cross-link same and to provide molecularflexibility. The linker preferably predominantly comprises amino acidswith small side chains, such as glycine, alanine and serine, to providefor flexibility. Preferably about 80 or 90 percent or greater of thelinker sequence comprises glycine, alanine or serine residues,particularly glycine and serine residues. For a fusion protein complexthat comprise a heterodimer TCR, the linker sequence is suitably linkedto the β chain of the TCR molecule, although the linker sequence alsocould be attached to the a chain of the TCR molecule. Alternatively,linker sequence may be linked to both α and β chains of the TCRmolecule. When such a β peptide chain is expressed along with the αchain, the linked TCR polypeptide should fold resulting in a functionalTCR molecule as generally depicted in FIG. 1. One suitable linkersequence is ASGGGGSGGG (i.e., Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly)(SEQ ID NO: 5), preferably linked to the first amino acid of the βdomain of the TCR. Different linker sequences could be used includingany of a number of flexible linker designs that have been usedsuccessfully to join antibody variable regions together, see Whitlow, M.et al., (1991) Methods: A Companion to Methods in Enzymology 2:97-105.In some examples, for covalently linking an effector molecule to a TCR βchain molecule, the amino sequence of the linker should be capable ofspanning suitable distance from the C-terminal residue of the TCR βchain to the N-terminal residue of the effector molecule. Suitablelinker sequences can be readily identified empirically. Additionally,suitable size and sequences of linker sequences also can be determinedby conventional computer modeling techniques based on the predicted sizeand shape of the TCR molecule.

T-Cell Receptors (TCR)

T-cells are a subgroup of cells which together with other immune celltypes (polymorphonuclear, eosinophils, basophils, mast cells, B-cells,NK cells), constitute the cellular component of the immune system. Underphysiological conditions T-cells function in immune surveillance and inthe elimination of foreign antigen. However, under pathologicalconditions there is compelling evidence that T-cells play a major rolein the causation and propagation of disease. In these disorders,breakdown of T-cell immunological tolerance, either central orperipheral is a fundamental process in the causation of autoimmunedisease.

The TCR complex is composed of at least seven transmembrane proteins.The disulfide-linked (αβ or γδ) heterodimer forms the monotypic antigenrecognition unit, while the invariant chains of CD3, consisting of ε, γ,δ, ζ, and η chains, are responsible for coupling the ligand binding tosignaling pathways that result in T-cell activation and the elaborationof the cellular immune responses. Despite the gene diversity of the TCRchains, two structural features are common to all known subunits. First,they are transmembrane proteins with a single transmembrane spanningdomain—presumably alpha-helical. Second, all TCR chains have the unusualfeature of possessing a charged amino acid within the predictedtransmembrane domain. The invariant chains have a single negativecharge, conserved between the mouse and human, and the variant chainspossess one (TCR-β) or two (TCR-α) positive charges. The transmembranesequence of TCR-α is highly conserved in a number of species and thusphylogenetically may serve an important functional role. The octapeptidesequence containing the hydrophilic amino acids arginine and lysine isidentical between the species.

A T-cell response is modulated by antigen binding to a TCR. One type ofTCR is a membrane bound heterodimer consisting of an α and β chainresembling an immunoglobulin variable (V) and constant (C) region. TheTCR α chain includes a covalently linked V-α and C-α chain, whereas theβ chain includes a V-β chain covalently linked to a C-β chain. The V-αand V-β chains form a pocket or cleft that can bind a superantigen orantigen in the context of a major histocompatibility complex (MHC)(known in humans as an HLA complex). See generally Davis Ann. Rev. ofImmunology 3: 537 (1985); Fundamental Immunology 3rd Ed., W. Paul Ed.Rsen Press LTD. New York (1993).

The extracellular domains of the TCR chains (αβ or γδ) can alsoengineered as fusions to heterologous transmembrane domains forexpression on the cell surface. Such TCRs may include fusions to CD3,CD28, CD8, 4-1BB and/or chimeric activation receptor (CAR) transmembraneor activation domains. TCRs can also be the soluble proteins comprisingone or more of the antigen binding domains of αβ or γδ chains. Such TCRsmay include the TCR variable domains or function fragments thereof withor without the TCR constant domains. Soluble TCRs may be heterodimericor single-chain molecules.

Recombinant Protein Expression

In general, preparation of the fusion protein complexes of the invention(e.g., components of Alt-803 or 2B8T2M) can be accomplished byprocedures disclosed herein and by recognized recombinant DNAtechniques.

In general, recombinant polypeptides are produced by transformation of asuitable host cell with all or part of a polypeptide-encoding nucleicacid molecule or fragment thereof in a suitable expression vehicle.Those skilled in the field of molecular biology will understand that anyof a wide variety of expression systems may be used to provide therecombinant protein. The precise host cell used is not critical to theinvention. A recombinant polypeptide may be produced in virtually anyeukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g.,Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, or preferably COScells). Such cells are available from a wide range of sources (e.g., theAmerican Type Culture Collection, Rockland, Md.; also, see, e.g.,Ausubel et al., Current Protocol in Molecular Biology, New York: JohnWiley and Sons, 1997). The method of transfection and the choice ofexpression vehicle will depend on the host system selected.Transformation methods are described, e.g., in Ausubel et al. (supra);expression vehicles may be chosen from those provided, e.g., in CloningVectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987).

A variety of expression systems exist for the production of recombinantpolypeptides. Expression vectors useful for producing such polypeptidesinclude, without limitation, chromosomal, episomal, and virus-derivedvectors, e.g., vectors derived from bacterial plasmids, frombacteriophage, from transposons, from yeast episomes, from insertionelements, from yeast chromosomal elements, from viruses such asbaculoviruses, papova viruses, such as SV40, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,and vectors derived from combinations thereof.

Once the recombinant polypeptide is expressed, it is isolated, e.g.,using affinity chromatography. In one example, an antibody (e.g.,produced as described herein) raised against the polypeptide may beattached to a column and used to isolate the recombinant polypeptide.Lysis and fractionation of polypeptide-harboring cells prior to affinitychromatography may be performed by standard methods (see, e.g., Ausubelet al., supra). Once isolated, the recombinant protein can, if desired,be further purified, e.g., by high performance liquid chromatography(see, e.g., Fisher, Laboratory Techniques In Biochemistry and MolecularBiology, eds., Work and Burdon, Elsevier, 1980).

As used herein, biologically active polypeptides or effector moleculesof the invention may include factors such as cytokines, chemokines,growth factors, protein toxins, immunoglobulin domains or otherbioactive proteins such as enzymes. Also biologically activepolypeptides may include conjugates to other compounds such asnon-protein toxins, cytotoxic agents, chemotherapeutic agents,detectable labels, radioactive materials and such.

Cytokines of the invention are defined by any factor produced by cellsthat affect other cells and are responsible for any of a number ofmultiple effects of cellular immunity. Examples of cytokines include butare not limited to the IL-2 family, interferon (IFN), IL-10, IL-1,IL-17, TGF and TNF cytokine families, and to IL-1 through IL-35, IFN-α,IFN-β, IFNγ, TGF-β, TNF-α, and TNFβ.

In an aspect of the invention, the first fusion protein comprises afirst biologically active polypeptide covalently linked tointerleukin-15 (IL-15) domain or a functional fragment thereof. IL-15 isa cytokine that affects T-cell activation and proliferation. IL-15activity in affecting immune cell activation and proliferation issimilar in some respects to IL2, although fundamental differences havebeen well characterized (Waldmann, T A, 2006, Nature Rev. Immunol.6:595-601).

In another aspect of the invention, the first fusion protein comprisesan interleukin-15 (IL-15) domain that is an IL-15 variant (also referredto herein as IL-15 mutant). The IL-15 variant preferably comprises adifferent amino acid sequence that the native (or wild type) IL-15protein. The IL-15 variant preferably binds the IL-15Rα polypeptide andfunctions as an IL-15 agonist or antagonist. Preferably IL-15 variantswith agonist activity have super agonist activity. In some embodiments,the IL-15 variant can function as an IL-15 agonist or antagonistindependent of its association with IL-15Rα. IL-15 agonists areexemplified by comparable or increased biological activity compared towild type IL-15. IL-15 antagonists are exemplified by decreasedbiological activity compared to wild type IL-15 or by the ability toinhibit IL-15-mediated responses. In some examples, the IL-15 variantbinds with increased or decreased activity to the IL-15RβγC receptors.In some embodiments, the sequence of the IL-15 variant has at least oneamino acid change, e.g. substitution or deletion, compared to the nativeIL-2 sequence, such changes resulting in IL-15 agonist or antagonistactivity. Preferably the amino acid substitutions/deletions are in thedomains of IL-15 that interact with IL-15Rβ and/or γC. More preferably,the amino acid substitutions/deletions do not affect binding to theIL-15Rα polypeptide or the ability to produce the IL-15 variant.Suitable amino acid substitutions/deletions to generate IL-15 variantscan be identified based on putative or known IL-15 structures,comparisons of IL-15 with homologous molecules such as IL-2 with knownstructure, through rational or random mutagenesis and functional assays,as provided herein, or other empirical methods. Additionally suitableamino acid substitutions can be conservative or non-conservative changesand insertions of additional amino acids. Preferably IL-15 variants ofthe invention contain one or more than one amino acidsubstitutions/deletions at position 6, 8, 10, 61, 65, 72, 92, 101, 104,105, 108, 109, 111, or 112 of the mature human IL-15 sequence;particularly, D8N (“D8” refers to the amino acid and residue position inthe native mature human IL-15 sequence and “N” refers to the substitutedamino acid residue at that position in the IL-15 variant), I6S, DBA,D61A, N65A, N72R, V104P or Q108A substitutions result in IL-15 variantswith antagonist activity and N72D substitutions result in IL-15 variantswith agonist activity.

Chemokines, similar to cytokines, are defined as any chemical factor ormolecule which when exposed to other cells are responsible for any of anumber of multiple effects of cellular immunity. Suitable chemokines mayinclude but are not limited to the CXC, CC, C, and CX₃C chemokinefamilies and to CCL-1 through CCL-28, CXC-1 through CXC-17, XCL-1,XCL-2, CX3CL1, MIP-1b, IL-8, MCP-1, and Rantes.

Growth factors include any molecules which when exposed to a particularcell induce proliferation and/or differentiation of the affected cell.Growth factors include proteins and chemical molecules, some of whichinclude: GM-CSF, G-CSF, human growth factor and stem cell growth factor.Additional growth factors may also be suitable for uses describedherein.

Toxins or cytotoxic agents include any substance that has a lethaleffect or an inhibitory effect on growth when exposed to cells. Morespecifically, the effector molecule can be a cell toxin of, e.g., plantor bacterial origin such as, e.g., diphtheria toxin (DT), shiga toxin,abrin, cholera toxin, ricin, saporin, pseudomonas exotoxin (PE),pokeweed antiviral protein, or gelonin. Biologically active fragments ofsuch toxins are well known in the art and include, e.g., DT A chain andricin A chain. Additionally, the toxin can be an agent active at thecell surface such as, e.g., phospholipase enzymes (e.g., phospholipaseC).

Further, the effector molecule can be a chemotherapeutic drug such as,e.g., vindesine, vincristine, vinblastin, methotrexate, adriamycin,bleomycin, or cisplatin.

Additionally, the effector molecule can be a detectably-labeled moleculesuitable for diagnostic or imaging studies. Such labels include biotinor streptavidin/avidin, a detectable nanoparticles or crystal, an enzymeor catalytically active fragment thereof, a fluorescent label such asgreen fluorescent protein, FITC, phycoerythrin, cychome, texas red orquantum dots; a radionuclide e.g., iodine-131, yttrium-90, rhenium-188or bismuth-212; a phosphorescent or chemiluminescent molecules or alabel detectable by PET, ultrasound or MRI such as Gd—or paramagneticmetal ion-based contrast agents. See e.g., Moskaug, et al. J. Biol.Chem. 264, 15709 (1989); Pastan, I. et al. Cell 47, 641, 1986; Pastan etal., Recombinant Toxins as Novel Therapeutic Agents, Ann. Rev. Biochem.61, 331, (1992); “Chimeric Toxins” Olsnes and Phil, Pharmac. Ther., 25,355 (1982); published PCT application no. WO 94/29350; published PCTapplication no. WO 94/04689; published PCT application no. WO2005046449and U.S. Pat. No. 5,620,939 for disclosure relating to making and usingproteins comprising effectors or tags.

A protein fusion or conjugate complex that includes a covalently linkedIL-15 and IL-15Rα domains has several important uses. For example, theprotein fusion or conjugate complex comprising a TCR can be employed todeliver the IL-15:IL-15Rα complex to certain cells capable ofspecifically binding the TCR. Accordingly, the protein fusion orconjugate complex provide means of selectively damaging or killing cellscomprising the ligand. Examples of cells or tissue capable of beingdamaged or killed by the protein fusion or conjugate complexescomprising a TCR include tumors and virally or bacterially infectedcells expressing one or more ligands capable of being specifically boundby the TCR. Cells or tissue susceptible to being damaged or killed canbe readily assayed by the methods disclosed herein.

The IL-15 and IL-15Rα polypeptides of the invention suitably correspondin amino acid sequence to naturally occurring IL-15 and IL-15Rαmolecules, e.g. IL-15 and IL-15Rα molecules of a human, mouse or otherrodent, or other mammal. Sequences of these polypeptides and encodingnucleic acids are known in the literature, including human interleukin15 (IL15) mRNA—GenBank: U14407.1, Mus musculus interleukin 15 (IL15)mRNA—GenBank: U14332.1, human interleukin-15 receptor alpha chainprecursor (IL15RA) mRNA—GenBank: U31628.1, Mus musculus interleukin 15receptor, alpha chain—GenBank: BC095982.1.

In some settings it can be useful to make the protein fusion orconjugate complexes of the present invention polyvalent, e.g., toincrease the valency of the sc-TCR or sc-antibody. In particular,interactions between the IL-15 and IL-15Rα domains of the fusion proteincomplex provide a means of generating polyvalent complexes. In addition,the polyvalent fusion protein can made by covalently or non-covalentlylinking together between one and four proteins (the same or different)by using e.g., standard biotin-streptavidin labeling techniques, or byconjugation to suitable solid supports such as latex beads. Chemicallycross-linked proteins (for example cross-linked to dendrimers) are alsosuitable polyvalent species. For example, the protein can be modified byincluding sequences encoding tag sequences that can be modified such asthe biotinylation BirA tag or amino acid residues with chemicallyreactive side chains such as Cys or His. Such amino acid tags orchemically reactive amino acids may be positioned in a variety ofpositions in the fusion protein, preferably distal to the active site ofthe biologically active polypeptide or effector molecule. For example,the C-terminus of a soluble fusion protein can be covalently linked to atag or other fused protein which includes such a reactive amino acid(s).Suitable side chains can be included to chemically link two or morefusion proteins to a suitable dendrimer or other nanoparticle to give amultivalent molecule. Dendrimers are synthetic chemical polymers thatcan have any one of a number of different functional groups of theirsurface (D. Tomalia, Aldrichimica Acta, 26:91:101 (1993)). Exemplarydendrimers for use in accordance with the present invention include e.g.E9 starburst polyamine dendrimer and E9 combust polyamine dendrimer,which can link cystine residues. Exemplary nanoparticles includeliposomes, core-shell particles or PLGA-based particles.

In another embodiment of the invention, one or both of the polypeptidesof the fusion protein complex comprises an immunoglobulin domain.Alternatively, the protein binding domain-IL-15 fusion protein can befurther linked to an immunoglobulin domain. The preferred immunoglobulindomains comprise regions that allow interaction with otherimmunoglobulin domains to form multichain proteins as provided above.For example, the immunoglobulin heavy chain regions, such as the IgG1C_(H)2-C_(H)3, are capable of stably interacting to create the Fcregion. Preferred immunoglobulin domains including Fc domains alsocomprise regions with effector functions, including Fc receptor orcomplement protein binding activity, and/or with glycosylation sites. Insome embodiments, the immunoglobulin domains of the fusion proteincomplex contain mutations that reduce or augment Fc receptor orcomplement binding activity or glycosylation, thereby affecting thebiological activity of the resulting protein. For example,immunoglobulin domains containing mutations that reduce binding to Fcreceptors could be used to generate fusion protein complex of theinvention with lower binding activity to Fc receptor-bearing cells,which may be advantageous for reagents designed to recognize or detectspecific antigens.

Nucleic Acids and Vectors

The invention further provides nucleic acid sequences and particularlyDNA sequences that encode the present fusion proteins (e.g., componentsof Alt-803 or 2B8T2M). Preferably, the DNA sequence is carried by avector suited for extrachromosomal replication such as a phage, virus,plasmid, phagemid, cosmid, YAC, or episome. In particular, a DNA vectorthat encodes a desired fusion protein can be used to facilitatepreparative methods described herein and to obtain significantquantities of the fusion protein. The DNA sequence can be inserted intoan appropriate expression vector, i.e., a vector that contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. A variety of host-vector systems may beutilized to express the protein-coding sequence. These include mammaliancell systems infected with virus (e.g., vaccinia virus, adenovirus,etc.); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors, or bacteriatransformed with bacteriophage DNA, plasmid DNA or cosmid DNA. Dependingon the host-vector system utilized, any one of a number of suitabletranscription and translation elements may be used. See generallySambrook et al., supra and Ausubel et al. supra.

Included in the invention are methods for making a soluble fusionprotein complex, the method comprising introducing into a host cell aDNA vector as described herein encoding the first and second fusionproteins, culturing the host cell in media under conditions sufficientto express the fusion proteins in the cell or the media and allowassociation between IL-15 domain of a first fusion protein and thesoluble IL-15Rα domain of a second fusion protein to form the solublefusion protein complex, purifying the soluble fusion protein complexfrom the host cells or media.

In general, a preferred DNA vector according to the invention comprisesa nucleotide sequence linked by phosphodiester bonds comprising, in a 5′to 3′ direction a first cloning site for introduction of a firstnucleotide sequence encoding a biologically active polypeptide,operatively linked to a sequence encoding an effector molecule.

The fusion protein components encoded by the DNA vector can be providedin a cassette format. By the term “cassette” is meant that eachcomponent can be readily substituted for another component by standardrecombinant methods. In particular, a DNA vector configured in acassette format is particularly desirable when the encoded fusioncomplex is to be used against pathogens that may have or have capacityto develop serotypes.

To make the vector coding for a fusion protein complex, the sequencecoding for the biologically active polypeptide is linked to a sequencecoding for the effector peptide by use of suitable ligases. DNA codingfor the presenting peptide can be obtained by isolating DNA from naturalsources such as from a suitable cell line or by known synthetic methods,e.g. the phosphate triester method. See, e.g., OligonucleotideSynthesis, IRL Press (M. J. Gait, ed., 1984). Synthetic oligonucleotidesalso may be prepared using commercially available automatedoligonucleotide synthesizers. Once isolated, the gene coding for thebiologically active polypeptide can be amplified by the polymerase chainreaction (PCR) or other means known in the art. Suitable PCR primers toamplify the biologically active polypeptide gene may add restrictionsites to the PCR product. The PCR product preferably includes splicesites for the effector peptide and leader sequences necessary for properexpression and secretion of the biologically active polypeptide-effectorfusion complex. The PCR product also preferably includes a sequencecoding for the linker sequence, or a restriction enzyme site forligation of such a sequence.

The fusion proteins described herein are preferably produced by standardrecombinant DNA techniques. For example, once a DNA molecule encodingthe biologically active polypeptide is isolated, sequence can be ligatedto another DNA molecule encoding the effector polypeptide. Thenucleotide sequence coding for a biologically active polypeptide may bedirectly joined to a DNA sequence coding for the effector peptide or,more typically, a DNA sequence coding for the linker sequence asdiscussed herein may be interposed between the sequence coding for thebiologically active polypeptide and the sequence coding for the effectorpeptide and joined using suitable ligases. The resultant hybrid DNAmolecule can be expressed in a suitable host cell to produce the fusionprotein complex. The DNA molecules are ligated to each other in a 5′ to3′ orientation such that, after ligation, the translational frame of theencoded polypeptides is not altered (i.e., the DNA molecules are ligatedto each other in-frame). The resulting DNA molecules encode an in-framefusion protein.

Other nucleotide sequences also can be included in the gene construct.For example, a promoter sequence, which controls expression of thesequence coding for the biologically active polypeptide fused to theeffector peptide, or a leader sequence, which directs the fusion proteinto the cell surface or the culture medium, can be included in theconstruct or present in the expression vector into which the constructis inserted. An immunoglobulin or CMV promoter is particularlypreferred.

In obtaining variant biologically active polypeptide, IL-15, IL-15Rα orFc domain coding sequences, those of ordinary skill in the art willrecognize that the polypeptides may be modified by certain amino acidsubstitutions, additions, deletions, and post-translationalmodifications, without loss or reduction of biological activity. Inparticular, it is well-known that conservative amino acid substitutions,that is, substitution of one amino acid for another amino acid ofsimilar size, charge, polarity and conformation, are unlikely tosignificantly alter protein function. The 20 standard amino acids thatare the constituents of proteins can be broadly categorized into fourgroups of conservative amino acids as follows: the nonpolar(hydrophobic) group includes alanine, isoleucine, leucine, methionine,phenylalanine, proline, tryptophan and valine; the polar (uncharged,neutral) group includes asparagine, cysteine, glutamine, glycine,serine, threonine and tyrosine; the positively charged (basic) groupcontains arginine, histidine and lysine; and the negatively charged(acidic) group contains aspartic acid and glutamic acid. Substitution ina protein of one amino acid for another within the same group isunlikely to have an adverse effect on the biological activity of theprotein. In other instance, modifications to amino acid positions can bemade to reduce or enhance the biological activity of the protein. Suchchanges can be introduced randomly or via site-specific mutations basedon known or presumed structural or functional properties of targetedresidue(s). Following expression of the variant protein, the changes inthe biological activity due to the modification can be readily assessedusing binding or functional assays.

Homology between nucleotide sequences can be determined by DNAhybridization analysis, wherein the stability of the double-stranded DNAhybrid is dependent on the extent of base pairing that occurs.Conditions of high temperature and/or low salt content reduce thestability of the hybrid, and can be varied to prevent annealing ofsequences having less than a selected degree of homology. For instance,for sequences with about 55% G-C content, hybridization and washconditions of 40-50 C, 6×SSC (sodium chloride/sodium citrate buffer) and0.1% SDS (sodium dodecyl sulfate) indicate about 60-70% homology,hybridization and wash conditions of 50-65 C, 1×SSC and 0.1% SDSindicate about 82-97% homology, and hybridization and wash conditions of52 C, 0.1×SSC and 0.1% SDS indicate about 99-100% homology. A wide rangeof computer programs for comparing nucleotide and amino acid sequences(and measuring the degree of homology) are also available, and a listproviding sources of both commercially available and free software isfound in Ausubel et al. (1999). Readily available sequence comparisonand multiple sequence alignment algorithms are, respectively, the BasicLocal Alignment Search Tool (BLAST) (Altschul et al., 1997) and ClustalWprograms. BLAST is available on the world wide web at ncbi.nlm.nih.govand a version of ClustalW is available at 2.ebi.ac.uk.

The components of the fusion protein can be organized in nearly anyorder provided each is capable of performing its intended function. Forexample, in one embodiment, the biologically active polypeptide issituated at the C or N terminal end of the effector molecule.

Preferred effector molecules of the invention will have sizes conduciveto the function for which those domains are intended. The effectormolecules of the invention can be made and fused to the biologicallyactive polypeptide by a variety of methods including well-known chemicalcross-linking methods. See e.g., Means, G. E. and Feeney, R. E. (1974)in Chemical Modification of Proteins, Holden-Day. See also, S. S. Wong(1991) in Chemistry of Protein Conjugation and Cross-Linking, CRC Press.However it is generally preferred to use recombinant manipulations tomake the in-frame fusion protein.

As noted, a fusion molecule or a conjugate molecule in accord with theinvention can be organized in several ways. In an exemplaryconfiguration, the C-terminus of the biologically active polypeptide isoperatively linked to the N-terminus of the effector molecule. Thatlinkage can be achieved by recombinant methods if desired. However, inanother configuration, the N-terminus of the biologically activepolypeptide is linked to the C-terminus of the effector molecule.

Alternatively, or in addition, one or more additional effector moleculescan be inserted into the biologically active polypeptide or conjugatecomplexes as needed.

Vectors and Expression

A number of strategies can be employed to express Alt-803 or 2B8T2M. Forexample, a construct encoding Alt-803 or 2B8T2M can be incorporated intoa suitable vector using restriction enzymes to make cuts in the vectorfor insertion of the construct followed by ligation. The vectorcontaining the gene construct is then introduced into a suitable hostfor expression of the fusion protein. See, generally, Sambrook et al.,supra. Selection of suitable vectors can be made empirically based onfactors relating to the cloning protocol. For example, the vector shouldbe compatible with, and have the proper replicon for the host that isbeing employed. Further the vector must be able to accommodate the DNAsequence coding for the fusion protein complex that is to be expressed.Suitable host cells include eukaryotic and prokaryotic cells, preferablythose cells that can be easily transformed and exhibit rapid growth inculture medium. Specifically preferred hosts cells include prokaryotessuch as E. coli, Bacillus subtillus, etc. and eukaryotes such as animalcells and yeast strains, e.g., S. cerevisiae. Mammalian cells aregenerally preferred, particularly J558, NSO, SP2-O or CHO. Othersuitable hosts include, e.g., insect cells such as Sf9. Conventionalculturing conditions are employed. See Sambrook, supra. Stabletransformed or transfected cell lines can then be selected. Cellsexpressing a fusion protein complex of the invention can be determinedby known procedures. For example, expression of a fusion protein complexlinked to an immunoglobulin can be determined by an ELISA specific forthe linked immunoglobulin and/or by immunoblotting. Other methods fordetecting expression of fusion proteins comprising biologically activepolypeptides linked to IL-15 or IL-15Rα domains are disclosed in theExamples.

As mentioned generally above, a host cell can be used for preparativepurposes to propagate nucleic acid encoding a desired fusion protein.Thus a host cell can include a prokaryotic or eukaryotic cell in whichproduction of the fusion protein is specifically intended. Thus hostcells specifically include yeast, fly, worm, plant, frog, mammaliancells and organs that are capable of propagating nucleic acid encodingthe fusion. Non-limiting examples of mammalian cell lines which can beused include CHO dhfr-cells (Urlaub and Chasm, Proc. Natl. Acad. Sci.USA, 77:4216 (1980)), 293 cells (Graham et al., J Gen. Virol., 36:59(1977)) or myeloma cells like SP2 or NSO (Galfre and Milstein, Meth.Enzymol., 73(B):3 (1981)).

Host cells capable of propagating nucleic acid encoding a desired fusionprotein comples encompass non-mammalian eukaryotic cells as well,including insect (e.g., Sp. frugiperda), yeast (e.g., S. cerevisiae, S.pombe, P. pastoris., K. lactis, H. polymorpha; as generally reviewed byFleer, R., Current Opinion in Biotechnology, 3(5):486496 (1992)), fungaland plant cells. Also contemplated are certain prokaryotes such as E.coli and Bacillus.

Nucleic acid encoding a desired fusion protein can be introduced into ahost cell by standard techniques for transfecting cells. The term“transfecting” or “transfection” is intended to encompass allconventional techniques for introducing nucleic acid into host cells,including calcium phosphate co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, electroporation, microinjection, viraltransduction and/or integration. Suitable methods for transfecting hostcells can be found in Sambrook et al. supra, and other laboratorytextbooks.

Various promoters (transcriptional initiation regulatory region) may beused according to the invention. The selection of the appropriatepromoter is dependent upon the proposed expression host. Promoters fromheterologous sources may be used as long as they are functional in thechosen host.

Promoter selection is also dependent upon the desired efficiency andlevel of peptide or protein production. Inducible promoters such as tacare often employed in order to dramatically increase the level ofprotein expression in E. coli. Overexpression of proteins may be harmfulto the host cells. Consequently, host cell growth may be limited. Theuse of inducible promoter systems allows the host cells to be cultivatedto acceptable densities prior to induction of gene expression, therebyfacilitating higher product yields.

Various signal sequences may be used according to the invention. Asignal sequence which is homologous to the biologically activepolypeptide coding sequence may be used. Alternatively, a signalsequence which has been selected or designed for efficient secretion andprocessing in the expression host may also be used. For example,suitable signal sequence/host cell pairs include the B. subtilis sacBsignal sequence for secretion in B. subtilis, and the Saccharomycescerevisiae α-mating factor or P. pastoris acid phosphatase phoI signalsequences for P. pastoris secretion. The signal sequence may be joineddirectly through the sequence encoding the signal peptidase cleavagesite to the protein coding sequence, or through a short nucleotidebridge consisting of usually fewer than ten codons, where the bridgeensures correct reading frame of the downstream TCR sequence.

Elements for enhancing transcription and translation have beenidentified for eukaryotic protein expression systems. For example,positioning the cauliflower mosaic virus (CaMV) promoter 1000 bp oneither side of a heterologous promoter may elevate transcriptionallevels by 10- to 400-fold in plant cells. The expression constructshould also include the appropriate translational initiation sequences.Modification of the expression construct to include a Kozak consensussequence for proper translational initiation may increase the level oftranslation by 10 fold.

A selective marker is often employed, which may be part of theexpression construct or separate from it (e.g., carried by theexpression vector), so that the marker may integrate at a site differentfrom the gene of interest. Examples include markers that conferresistance to antibiotics (e.g., bla confers resistance to ampicillinfor E. coli host cells, nptII confers kanamycin resistance to a widevariety of prokaryotic and eukaryotic cells) or that permit the host togrow on minimal medium (e.g., HIS4 enables P. pastoris or His⁻ S.cerevisiae to grow in the absence of histidine). The selectable markerhas its own transcriptional and translational initiation and terminationregulatory regions to allow for independent expression of the marker. Ifantibiotic resistance is employed as a marker, the concentration of theantibiotic for selection will vary depending upon the antibiotic,generally ranging from 10 to 600 μg of the antibiotic/mL of medium.

The expression construct is assembled by employing known recombinant DNAtechniques (Sambrook et al., 1989; Ausubel et al., 1999). Restrictionenzyme digestion and ligation are the basic steps employed to join twofragments of DNA. The ends of the DNA fragment may require modificationprior to ligation, and this may be accomplished by filling in overhangs,deleting terminal portions of the fragment(s) with nucleases (e.g.,ExoIII), site directed mutagenesis, or by adding new base pairs by PCR.Polylinkers and adaptors may be employed to facilitate joining ofselected fragments. The expression construct is typically assembled instages employing rounds of restriction, ligation, and transformation ofE. coli. Numerous cloning vectors suitable for construction of theexpression construct are known in the art (λZAP and pBLUESCRIPT SK-1,Stratagene, La Jolla, Calif., pET, Novagen Inc., Madison, Wis., cited inAusubel et al., 1999) and the particular choice is not critical to theinvention. The selection of cloning vector will be influenced by thegene transfer system selected for introduction of the expressionconstruct into the host cell. At the end of each stage, the resultingconstruct may be analyzed by restriction, DNA sequence, hybridizationand PCR analyses.

The expression construct may be transformed into the host as the cloningvector construct, either linear or circular, or may be removed from thecloning vector and used as is or introduced onto a delivery vector. Thedelivery vector facilitates the introduction and maintenance of theexpression construct in the selected host cell type. The expressionconstruct is introduced into the host cells by any of a number of knowngene transfer systems (e.g., natural competence, chemically mediatedtransformation, protoplast transformation, electroporation, biolistictransformation, transfection, or conjugation) (Ausubel et al., 1999;Sambrook et al., 1989). The gene transfer system selected depends uponthe host cells and vector systems used.

For instance, the expression construct can be introduced into S.cerevisiae cells by protoplast transformation or electroporation.Electroporation of S. cerevisiae is readily accomplished, and yieldstransformation efficiencies comparable to spheroplast transformation.

The present invention further provides a production process forisolating a fusion protein of interest. In the process, a host cell(e.g., a yeast, fungus, insect, bacterial or animal cell), into whichhas been introduced a nucleic acid encoding the protein of the interestoperatively linked to a regulatory sequence, is grown at productionscale in a culture medium to stimulate transcription of the nucleotidessequence encoding the fusion protein of interest. Subsequently, thefusion protein of interest is isolated from harvested host cells or fromthe culture medium. Standard protein purification techniques can be usedto isolate the protein of interest from the medium or from the harvestedcells. In particular, the purification techniques can be used to expressand purify a desired fusion protein on a large-scale (i.e. in at leastmilligram quantities) from a variety of implementations including rollerbottles, spinner flasks, tissue culture plates, bioreactor, or afermentor.

An expressed protein fusion complex can be isolated and purified byknown methods. Typically the culture medium is centrifuged or filteredand then the supernatant is purified by affinity or immunoaffinitychromatography, e.g. Protein-A or Protein-G affinity chromatography oran immunoaffinity protocol comprising use of monoclonal antibodies thatbind the expressed fusion complex such as a linked TCR or immunoglobulinregion thereof. The fusion proteins of the present invention can beseparated and purified by appropriate combination of known techniques.These methods include, for example, methods utilizing solubility such assalt precipitation and solvent precipitation, methods utilizing thedifference in molecular weight such as dialysis, ultra-filtration,gel-filtration, and SDS-polyacrylamide gel electrophoresis, methodsutilizing a difference in electrical charge such as ion-exchange columnchromatography, methods utilizing specific affinity such as affinitychromatography, methods utilizing a difference in hydrophobicity such asreverse-phase high performance liquid chromatography and methodsutilizing a difference in isoelectric point, such as isoelectricfocusing electrophoresis, metal affinity columns such as Ni-NTA. Seegenerally Sambrook et al. and Ausubel et al. supra for disclosurerelating to these methods.

It is preferred that the fusion proteins of the present invention besubstantially pure. That is, the fusion proteins have been isolated fromcell substituents that naturally accompany it so that the fusionproteins are present preferably in at least 80% or 90% to 95%homogeneity (w/w). Fusion proteins having at least 98 to 99% homogeneity(w/w) are most preferred for many pharmaceutical, clinical and researchapplications. Once substantially purified the fusion protein should besubstantially free of contaminants for therapeutic applications. Oncepurified partially or to substantial purity, the soluble fusion proteinscan be used therapeutically, or in performing in vitro or in vivo assaysas disclosed herein. Substantial purity can be determined by a varietyof standard techniques such as chromatography and gel electrophoresis.

In certain embodiments, soluble TCR fusion complexes of the inventioncontain TCR domains that is sufficiently truncated so the TCR fusioncomplex can be secreted into culture medium after expression. Thus, atruncated TCR fusion complex will not include regions rich inhydrophobic residues, typically the transmembrane and cytoplasmicdomains of the TCR molecule. Thus, for example, for a preferredtruncated TCR molecule of the invention, preferably from about the finalcysteine to the C-terminal residue of the β chain and from about thefinal cysteine to the C-terminal residue of the α chain of the TCRmolecule are not included in the truncated TCR fusion complex.

The present fusion protein complexes are suitable for in vitro or invivo use with a variety of cells that are cancerous or are infected orthat may become infected by one or more diseases.

Human interleukin-15 (hIL-15) is trans-presented to immune effectorcells by the human IL-15 receptor α chain (hIL-15Rα) expressed onantigen presenting cells. IL-15Rα binds hIL-15 with high affinity (38pM) primarily through the extracellular sushi domain (hIL-15RαSu. ThehIL-15 and hIL-15RαSu domains can be used as a scaffold to constructmulti-domain fusion complexes. Both bivalent and bispecific T cellreceptor (TCR) fusion complexes were formed using this scaffold throughthe combination of various single-chain (sc) TCR domains fused to theN-termini of the hIL-15 and hIL-15RαSu chains. In these fusions, thescTCR domains retain the antigen binding activity and the hIL-15 domainexhibits receptor binding and biological activity. Bivalent scTCRfusions exhibited improved antigen binding capacity due to increasedmolecular binding avidity whereas fusions comprising two different scTCRdomains were capable of binding two cognate peptide/MHC complexes.Bispecific molecules containing scTCR and scCD8αβ domains also exhibitsignificantly better binding to cognate peptide/MHC complex than eitherthe bivalent or monovalent scTCR molecules, demonstrating that theIL-15:IL-15Rα scaffold exhibits flexibility necessary to supportmulti-domain interactions with given target. Surprisingly, functionalTCRs could also be formed by co-expressing the TCR α and β chainsseparately as fusions to the hIL-15 and hIL-15RαSu domains. Finally, thefused hIL-15 domain can be manipulated through site-specific mutationsto provide superagonist or antagonist cytokine activity. Together, theseproperties indicate that the hIL-15 and hIL-15RαSu domains can be usedas versatile, functional scaffold for generating novel targeted immunemolecules.

IgG domains, particularly the Fc fragment, have been used successfullyas dimeric scaffolds for a number of therapeutic molecules includingapproved biologic drugs. For example, etanercept is a dimer of solublehuman p75 tumor necrosis factor-α (TNF-α) receptor (sTNFR) linked to theFc domain of human IgG1. This dimerization allows etanercept to be up to1,000 times more potent at inhibiting TNF-α activity than the monomericsTNFR and provides the fusion with a five-fold longer serum half-lifethan the monomeric form. As a result, etanercept is effective atneutralization of the pro-inflammatory activity of TNF-α in vivo andimproving patient outcomes for a number of different autoimmuneindications.

In addition to its dimerization activity, the Fc fragment also providescytotoxic effector functions through the complement activation andinteraction with Fcγ receptors displayed on natural killer (NK) cells,neutrophils, phagocytes and dendritic cells. In the context ofanti-cancer therapeutic antibodies and other antibody domain-Fc fusionproteins, these activities likely play an important role in efficacyobserved in animal tumor models and in cancer patients. However thesecytotoxic effector responses may not be sufficient in a number oftherapeutic applications. Thus, there has been considerable interest inimproving and expanding on the effector activity of the Fc domain anddeveloping other means of recruiting cytolytic immune responses,including T cell activity, to the disease site via targeted therapeuticmolecules. IgG domains have been used as a scaffold to form bispecificantibodies to improve the quality and quantity of products generated bythe traditional hybridoma fusion technology. Although these methodsbypass the shortcomings of other scaffolds, it has been difficult toproduce bispecific antibodies in mammalian cells at levels sufficient tosupport clinical development and use.

In an effort to develop a new, human-derived immunostimulatorymultimeric scaffold, human IL-15 (hIL-15) and IL-15 receptor domainswere used. hIL-15 is a member of the small four α-helix bundle family ofcytokines that associates with the hIL-15 receptor α-chain (hIL-15RαγC)with a high binding affinity (Equilibrium dissociation constant (KD)˜10⁻¹¹ M). The resulting complex is then trans-presented to the humanIL-2/15 receptor (β/common γ chain (hIL-15RβγC) complexes displayed onthe surface of T cells and NK cells. This cytokine/receptor interactionresults in expansion and activation of effector T cells and NK cells,which play an important role in eradicating virally infected andmalignant cells. Normally, hIL-15 and hIL-15Rα are co-produced indendritic cells to form complexes intracellularly that are subsequentlysecreted and displayed as heterodimeric molecules on cell surfaces.Thus, the characteristics of hIL-15 and hIL-15Rα interactions suggestthat these inter chain binding domains could serve as a novel,human-derived immunostimulatory scaffold to make soluble dimericmolecules capable of target-specific binding. A number of fusionproteins comprising T cell receptor (TCR) and CD8 binding domains weremade to demonstrate the feasibility of using hIL-15:hIL-15Rα scaffold tocreate both soluble homodimers with increased functional bindingaffinity toward target antigens and heterodimers formultiple-site-specific protein complexes. These fusion proteins retainpotent hIL-15 activity capable of stimulating immune effector cellresponses.

An hIL-15:hIL-15RαSu-based scaffold was used to create novel, dimericmolecules. The dimeric fusion protein complexes retainedimmunostimulatory and target-specific biological activity of theirhIL-15 domains and binding domains, indicating that the addition ofhIL-15 and hIL-15Rα did not significantly alter the spatial arrangementof the fusion domains and provided an adequate degree of conformationalflexibility without impacting cytokine activity. Thus, this scaffoldcould be used to form multivalent fusion complexes, such as thec264scTCR dimer, to increase the overall binding affinity of molecules,or bispecific molecules, such as the c264scTCR/c149scTCR heterodimer. Inall cases, the soluble fusion proteins were produced at relatively highlevels in recombinant CHO cell culture (mgs per liter in cell culturesupernatant without extensive cell line screening or optimization) andcould be readily purified from the cell culture supernatants. ThehIL-15:hIL-15RαSu-based scaffold could be expanded to create soluble,biologically active, two-chain molecules, such as α/β TCRs, by fusingthe extracellular domains of the two polypeptide chains to the N-terminiof hIL-15 and hIL-15RαSu. This format resulted in a moderate decrease inhIL-15 activity, possibly due to steric hindrance between theinterfolded TCR α/β chains fused to the distal N-termini of thehIL-15:hIL-15RαSu complex and the hIL-15RβγC binding site located in themiddle of the complex. Other formats are possible and can be generatedusing routine methods.

The hIL-15:hIL-15RαSu-based scaffold was also used to generate anOT1scTCR/scCD8 heterodimer in which the CD8 α/β and TCR domains arecapable of binding the same pMHCI complex but at a spatially distinctsites. Previous studies using soluble pMHCI reagents have determinedthat CD8 stabilizes and enhances TCR:pMHCI interactions at the cellsurface through effects on both the off-rate and the on-rate. Thiseffect is important in determining the dependency of the T cells on CD8co-receptor activity, such that the requirement for CD8 forpMHCI-specific T cell activation is inversely correlated with TCR:pMHCIaffinity. However, several binding studies using soluble purified CD8α/β, TCR and pMHCI proteins have shown that TCR:pMHCI interactions arenot affected by the presence or absence of CD8, suggesting nocooperative binding effects.

The results of cell-based and SPR binding studies with theOT1scTCR/scCD8 heterodimer are in contrast with earlier reports showingthat TCR and CD8 domains displayed on the same soluble moleculeexhibited much better peptide/MHC binding activity than was observedwith molecules carrying monovalent or divalent TCR domains. This effectis reflected in both a slower off-rate and faster on-rate of thepMHCI:OT1scTCR/scCD8 heterodimer complex, consistent with theobservations for pMHCI binding to CD8 and TCR molecules on T cells.Thus, the OT1scTCR/scCD8 heterodimer mimics binding of the OT1 TCR on Tcells, which exhibits a strong dependence of CD8 coreceptor activity forpMHC interactions. These results indicate that the scTCR/scCD8heterodimer and variants of this molecule could serve as very usefultools for further dissecting molecular interactions between the tertiaryTCR:pMHCI:CD8 complex in a cell-free system. In addition, scTCR/scCD8heterodimer-based reagents with enhanced pMHCI binding activity couldhave utility in detecting antigen presentation on diseased cells,without the need of mutating the TCR domain for increased bindingaffinity.

The results of SPR experiments on the OT1scTCR fusions differ from thosereported by Alam et al. where the binding affinity of monovalent OT1TCRα/β heterodimer to immobilized OVA peptide/H-2Kb complex was shown tobe approximately 6 μM. In our studies, we were unable to detect OVApeptide/H-2Kb-binding of the OT1scTCR/birA monomer and the OT1scTCR/birAdimer exhibited an apparent KD of 30 μM. It is possible that the OT1 TCRlost binding activity when formatted as a single-chain Vα-linker-Vβ-Cβmolecule. However, we observed equivalent activity when comparingOT1scTCR/birA and a two-chain construct. Moreover, previous studies haveshown that OVA peptide/H-2 Kb tetramers with Kb mutations that abrogateCD8 binding exhibit little or no specific binding activity to OT1TCR-bearing cells even when high concentrations of tetramers were used,suggesting very low affinity interactions between OT1 TCR and itscognate pMHCI. In contrast, OVA peptide/H-2 Kb tetramers without the CD8binding mutations were able to brightly stain OT1 TCR-bearing cells,consistent with the ability of CD8 to enhance OT1 TCR binding activityobserved in this study.

The hIL-15:hIL-15RαSu-based scaffold can be exploited much like the Fcdomain of the IgG scaffold to generate multivalent or multispecifictargeted therapeutics. With its potent activity for stimulatingproliferation and activation of effector NK and CD8⁺ memory T cells, thehIL-15 domain expands the scope of potential immunotherapeuticmechanisms beyond antibody-dependent cellular cytotoxicity andcomplement activation associated with IgG-based approaches. Usingapproaches similar to those used to manipulate the activity of the Fcdomain of IgG molecules, we demonstrate that the IL-15 domain can bemutated to increase or decrease its functional activity. We show thathIL-15:hIL-15RαSu fusion molecule containing an N72D mutation in theIL-15 domain exhibit a 3-4 fold increase in biological activity, whereasIL-15 D8N mutation exhibit little or no activity. While IL-15superagonist-based fusion proteins could serve as targetedimmunotherapeutics for cancer and infectious diseases, an IL-15antagonist capable of inhibiting IL-15 responsive cells at the diseasesite may have therapeutic potential in treating allograft rejection andinflammatory autoimmune diseases, particularly if memory CD8 T cellsplay a role in disease pathology. A non-targeted IL-15 mutant/Fcγ2aantagonist protein has already been shown to be effective at inhibitingislet and cardiac allograft rejection and preventing development andprogression of arthritis in experimental animal models. Similarapproaches with IL-15 antagonist domains in the context of thehIL-15:hIL-15RαSu fusion proteins are possible. In addition, undercertain circumstances, it may desirable to have a functionally inertscaffold for generation of multimeric molecules. For example, we havefound that scTCR/hIL-15:scTCR/hIL-15RαSu fusions containing an IL-15 D8Nmutation, which eliminates interactions with IL-15Rβγ, provide betterTCR antigen-specific staining of cells displaying IL-15 receptorcomplex.

Although TCRs and CD8 molecules were used as targeting domains fordemonstration purposes herein, it is understood that thehIL-15:hIL-15RαSu scaffold could be used to construct other novelmolecules with protein domains derived from antibodies, adhesionmolecules, or other receptors. It is also possible to create proteindomain fusions to the C-termini of the hIL-15 and hIL-15RαSu which,based on the crystal structure, are accessible for modification. Theresulting molecules can contain up to four different target-recognitioncapabilities. With the appropriate fusion partners, these types ofmolecules can promote the conjugation of immune effectors cells andtarget cells and achieve effective killing of target cells. In addition,the IL-15 domain of the complex can further augment these processes byproviding immunostimulatory activity to support effector cellproliferation and cytotoxicity. A variety of multi-functional moleculesbased on this concept for use as anti-cancer and anti-viralimmunotherapeutic agents.

Previously, the poor expression level in standard mammalian cell systemlimited the development of recombinant hIL-15 as a therapeutic. Asdemonstrated herein, expression of scTCR/hIL-15:scTCR/hIL-15RαSucomplexes at levels capable of supporting clinical development andpotentially product commercialization can be achieved. In addition, ithas been shown that the IL-15Rα chain enhances the in vivo activity ofhIL-15, without being bound by mechanism, possibly by improving thepharmacokinetics of the cytokine. These two characteristics ofhIL-15:hIL-15RαSu complexes, in combination with its multivalent natureand/or multispecific targeting design, provides an opportunity tocapture the full potential of hIL-15 as an immunotherapeutic agentagainst cancer and viral infections.

As provided in the Examples, hIL-15:hIL-15RαSu fusion protein complexescomprising immunoglobulin Fc domains were found to have additionaladvantages. Association of the Fc domains allows generation ofmultichain molecules capable of multivalent and multispecificinteractions. In fact, the fusion protein complexes of the inventioncomprising the multiple domains of the same scTCR exhibited enhancedantigen binding activity than that expected based on the activity of thedimeric scTCR fusion. In some cases, the fusion complex of the inventionis capable of binding and activating both IL-15RβγC-bearing immune cellsand Fc receptor-bearing immune cells, allowing for potent immunestimulatory activity. The protein fusion complex of the inventioncomprising two IL-15 domains was found to exhibited better IL-15activity than that expected when compared to other IL-15 fusionproteins. Additionally, the protein fusion complex of the invention wasmore effective at mediating antibody Fc depended cellular cytotoxicityagainst peptide/MHC presenting target cells than the TCR-IgG1 fusionprotein. The improved activity may have been the result of enhancedbinding of the protein fusion complexes to the peptide/MHC complexand/or increase reactivity to the effector cells displaying Fc receptorsor IL-15 receptors. Moreover, through mutagenesis analysis it was foundthat of each of the TCR, IL-15 and IgG Fc domains of the fusion proteincomplexes could be readily and independently manipulated to alter itsbinding and functional activity to provide a multispecific complex withthe desired biological effects.

The fusion protein complexes of the invention were demonstrated to havea significantly better pharmacokinetic profile in mammals than freeIL-15. In addition, based on the similar PK profile observed withdifferent methods of analysis, the fusion protein complexes remainsintact in vivo as a multichain molecule with no evidence of polypeptidechain cleavage or dissociation. Additionally, the fusion proteincomplexes of the invention are shown to be capable of mediatingantitumor activity against both target bearing and non-target bearingtumors in animals and exhibited more potent antitumor efficacy thanrhIL-15 administered at an equivalent molar dose. Moreover, treatmentwith effective doses of the fusion proteins was well tolerated in theseanimal models.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES Example 1 Construction of Expression Vectors Containingc264scTCR/huIL15RαSushi-huIgG1 and c149scTCR/huIL15N72D Gene Fusions

The fusion protein referred to as the T2 molecule (T2M) consists of amultichain polypeptide (FIG. 1). In one embodiment of the invention, oneof these polypeptides comprises a fusion between a protein-bindingdomain and IL-15 (or IL-15 variants) as disclosed in WO2008143794(incorporated herein by reference). A second polypeptide of T2 comprisesa fusion between a protein binding domain, an IL-15Rα domain and animmunoglobulin domain. Alternatively, the protein binding domain-IL-15fusion protein can be further linked to an immunoglobulin domain. Thepreferred immunoglobulin domains comprise regions that allow interactionwith other immunoglobulin domains to form multichain proteins. Forexample, the immunoglobulin heavy chain regions, such as the IgG1C_(H)2-C_(H)3, are capable of interacting to create the Fc region.Preferred immunoglobulin domains also comprise regions with effectorfunctions, including Fc receptor or complement protein binding activity,and/or with glycosylation sites. In some embodiments, the immunoglobulindomains of the T2 molecule contain mutations that reduce or augment Fcreceptor or complement binding activity or glycosylation, therebyaffecting the biological activity of the resulting protein. For example,immunoglobulin domains containing mutations that reduce binding to Fcreceptors could be used to generate T2 molecules with lower bindingactivity to Fc receptor-bearing cells, which may be advantageous forreagents designed to recognize or detect TCR-specific antigens.

Construction of an expression vector containing the p53 (aa 264-272)single-chain TCR (referred to a c264scTCR) fused to human IL-15Rα sushidomain (huIL15RαSushi) and human IgG1 constant regions (huIgG1C_(H)1-C_(H)2-C_(H)3) was carried out as follows. The c264scTCR/huIgG1gene fragment was removed from the previous constructed thepNEF38-c264scTCR/huIgG1 vector by restricted digestion with PacI andM1uI. The gene fragment was gel-purified and ligated to pMSGV vectordigested with the same restriction enzymes, resulted in the constructcalled as pMSGV-c264scTCR/huIgG1. A DNA fragment containing the CMVpromoter was purified from pcDNA3.1 following digestion with NruI andHindIII. This fragment was ligated into pMSGV-c264scTCR/huIgG1 which hadbeen digested with PacI and filled in with DNA polymerase to createblunt ends and then digested with HindIII. The resulting construct wasnamed as pMC-c264scTCR/huIgG1. A huIL15RαSushi gene fragment from aprevious constructed, pNEF38-c264scTCR/huIL15RαSushi (see WO2008143794),was amplified with front primer:

(SEQ ID NO: 6) 5′-TGTTGGGAATTCATCACGTGCCCTC-3′and back primer:

(SEQ ID NO: 7) 5′-TGGTGTGAATTCTCTAATGCATTTGAGACTGG-3′by KOD Hot Start DNA Polymerase (EMD) under following PCR conditions:95C, 2 min, 1 cycle; 95 C, 20 sec, 65 C, 20 sec; 70 C, 20 sec, 35cycles; 72C, 10 min, 1 cycle. The PCR product of human IL15RαSushi genewas gel-purified and digested with EcoRI. The gene was ligated intopMC-c264scTCR/huIgG1 which had been digested with EcoRI. Cloning of theDNA fragment encoding the human IL15RαSushi domain into thepMC-c264scTCR/huIgG1 resulted in a c264scTCR/huIL15RαSushi-huIgG1 fusiongene comprising the following sequence: 3′-immunoglobulin heavy chainleader-264 TCR V-α—peptide linker-264 TCR V-β—human TCR C-β—humanIL15RαSushi—human IgG1 heavy chain. The resulting vector(pMC.c264scTCR-Su/IgG1.PUR), shown in FIG. 2, containing the correcthuman IL15RαSushi gene insert was identified based on the diagnostic PCRand reconfirmed by DNA sequencing. The sequences of thec264scTCR/huIL15RαSushi/huIgG1 gene and protein are shown at FIGS. 3A-3Cand FIGS. 4A-4D, respectively.

A different expression vector containing c264scTCR/huIL15RαSushi-huIgG1gene fusion was constructed that lacked the internal EcoRI sites (andcorresponding coding sequences). For this vector, a portion of thec264scTCR gene fragment was amplified from the c264scTCR/huIgG1 vectorwith front primer:

(SEQ ID NO: 8) 5′GTACGACTTAATTAACTCGAGCCACCATGGAGACAGACACACTCCTGTTATGG3′and back primer:

(SEQ ID NO: 9) 5′CTTCCCGTTAACCCACCAGCTCAGCTCCACGTG3′.

The remainder of the TCR β constant region of the c264scTCR genefragment was amplified from c264scTCR/huIgG1 vector with front primer:

(SEQ ID NO: 10) 5′CTGGTGGGTTAACGGGAAGGAGGTGCACAGTGGGGTC3′and back primer:

(SEQ ID NO: 11) 5′GAGGGCACGTGATGTCTGCTCTACCCCAGGCCTC3′

The huIL15RαSushi gene fragment was amplified from thec264scTCR/huIL15RαSushi vector with front primer:

(SEQ ID NO: 12) 5′GTAGAGCAGACATCACGTGCCCTCCCCCCATG3′and the back primer:

(SEQ ID NO: 13) 5′CCTTGGTGCTAGCTCTAATACATTTGAGACTGGGGGTTGTCC3′.

The huIgG1 heavy chain constant region gene fragment was amplified fromthe c264scTCR/huIgG1 vector with front primer:

(SEQ ID NO: 14) 5′CCAGTCTCAAATGTATTAGAGCTAGCACCAAGGGCCCATCGGTC3′and back primer:

(SEQ ID NO: 15) 5′GTAATATTCTAGACGCGTTCATTATTTACCAGGAGACAGGGAGAGGCTCTTC3′.

The resulting products containing the TCR β constant region sequence andhuIL15RαSushi gene were used as templates to generate a gene fragment byPCR using with front primer:

(SEQ ID NO: 10) 5′CTGGTGGGTTAACGGGAAGGAGGTGCACAGTGGGGTC3′and back primer:

(SEQ ID NO: 15) 5′CCTTGGTGCTAGCTCTAATACATTTGAGACTGGGGGTTGTC C3′

The resulting PCR product and the huIgG1 gene fragment served astemplates to generate a TCRβc/huIL15RαSushi/huIgG1 fusion gene by PCRwith front primer:

5′CTGGTGGGTTAACGGGAAGGAGGTGCACAGTGGGGTC3′and back primer:

5′GTAATATTCTAGACGCGTTCATTATTTACCAGGAGACAGGGA GAGGCTCTTC3′

The c264scTCR PCR product was digested with PacI and HpaI and theTCRβc/huIL15RαSushi/huIgG1 fusion gene was digested with HpaI and NsiI.The digested gene fragments were ligated into a CMV promoter-containingpMSGV retrovirus vector. The resulting vector was designated asc264scTCR/Sushi/hIgG1-pMSGVc or pMSGVc264SuIg (FIG. 5). The sequences ofthe c264scTCR/huIL15RαSushi/huIgG1 gene and protein are shown at FIG.6A, FIG. 6B, and FIG. 6C and FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D,respectively.

Generation of expression vectors producing a fusion between single-chainTCR binding domain (i.e. c264scTCR) and IL-15 (or IL-15 variants) hasbeen disclosed in WO2008143794. Particularly useful IL-15 variants arethose that reduce or eliminate IL-15 biological activity or thatincrease IL-15 biological activity. For example, human IL-15 variantswith substitutions at position 72 (i.e. N72D substitution) can increasethe IL-15 biological activity 5 to 10 fold. IL-15 variants are providedin the table below:

IL15Rβγc receptor IL15Rα Proliferation Mutants Position 8 61 65 72 108binding binding Activity WTaa D D N N Q + + + 1 8 N − + − 2 8 A − + − 361 A − + − 4 65 D − + − 5 65 A − + − 6 72 D 3+ + 3+ 8 72 R − + − 9 108 A− + − 10  8 + 65 N A − + − 11   8 + 108 A A − + − 12  8 + 65 S R − + −

The fusion protein complexes comprising IL-15 variants as described inthe table immediately above were characterized for their ability to bindthe TCR-specific antigen, p53 (aa264-272)/HLA-A2.1. To generate cellspresenting p53 (aa264-272)/HLA-A2.1, HLA-A2.1-positive T2 cells(2×10⁶/mL) were loaded with 20 μM p53 (aa264-272) peptide at 37° C. inthe presence of 1×PLE (Altor Bioscience) for 2-3 hrs. T2 cells that werenot incubated with peptide and 32Dβ cells expressing IL-2/15Rβγ_(C)serve as controls. The p53 peptide-loaded T2 cells, control T2 cells, or32Dβ cells (2×10⁵/100 μL) were then incubated for 30 min at 4 C with 320nM of following dimeric fusion protein complexes: 1)c264scTCR/huIL15+c264scTCR/huIL15Rα Sushi, 2)c264scTCR/huIL15D8A+c264scTCR/huIL15Rα Sushi, and 3)c264scTCR/huIL15D8N+c264scTCR/huIL15Rα Sushi. These complexes weregenerated by incubating 160 nM of purified c264scTCRhuIL15 fusionprotein and 160 nM of purified c264scTCRhuIL15Rα Sushi fusion protein at4 C for 3 hours. Following staining, cells were washed once with washingbuffer (PBS containing 0.5% BSA and 0.05% sodium azide) and stained with0.5 μg of biotinylated mouse monoclonal anti-human TCR Cβ antibody (BF1)in 100 μL of washing buffer for 30 min at 4 C. Cells were washed onceand stained with 0.5 μg of R-Phycoerythrin conjugated streptavidin in100 μL of washing buffer for 30 min at 4 C. Cells were washed andresuspended for analysis by flow cytometry.

The c264scTCR/huIL15D8A+c264scTCR/huIL15RαSushi complex andc264scTCR/huIL15D8N+c264scTCR/huIL15RαSushi complex exhibited equivalentactivity as the c264scTCR/huIL15+c264scTCR/huIL15RαSushi complex forspecifically staining p53 peptide-loaded T2 cells. These resultsindicate that the multivalent scTCR domains are fully functional in eachof these fusion complexes. Fusion protein complexes comprising IL-15variants (D8A and D8N) do not show binding activity to the IL-15Rβγ_(c)receptors present on the 32Dβ cells. Similar studies of IL-15Rβγ_(c)receptor binding were carried out with other fusion proteins comprisingIL-15 variants and are summarized in Table 1. The results indicate thatfusion proteins and fusion protein complexes of the invention comprisingIL-15 variants retain activity to recognize peptide/MHC complexes andexhibit decreased or increased binding activity for IL-15Rβγ_(c)receptors.

For certain T2 molecules, it is useful to have multiple differentbinding domains fused to the IL-15 and IL-15Rα components. In oneexample to illustrate the activity of such molecules, a single-chain TCRdomain (called c149scTCR), specific to the p53 (aa 149-157) peptidepresented in the context of HLA-A2, was linked to the IL-15N72D domainand the resulting fusion protein co-expressed with thec264scTCR/huIL15RαSushi/huIgG1 fusion protein to produce a multichain T2protein with c264scTCR and c149scTCR binding domains.

To generate the c149scTCR/IL15N72D gene fusion, a c149scTCR genefragment (TCR-α, linker, TCR-β and TCR-β constant fragment) wasamplified from c149scTCR/huIgG1 expression vector with the front primer:

(SEQ ID NO: 16) 5′GACTTCAAGCTTAATTAAGCCACCATGGACAGACTTACTTCT TC3′and the back primer:

(SEQ ID NO: 9) 5′-CTTCCCGTTAACCCACCAGCTCAGCTCCACGTG-3′

The remainder of the TCR β constant region of the c149scTCR/huIgG1vector was amplified with front primer:

(SEQ ID NO: 17) 5′CTGGTGGGTTAACGGGAAGGAGGTGCACAGTGGGGTC3′and the back primer:

(SEQ ID NO: 18) 5′CACCCAGTTGTCTGCTCTACCCCAGGCCTC3′

The huIL15N72D gene was amplified from c264scTCR/huIL15N72D expressionvector with the front primer:

(SEQ ID NO: 19) 5′CTGGGGTAGAGCAGACAACTGGGTGAATGTAATAAGTGATTT G3′and the back primer:

(SEQ ID NO: 20) 5′CCTCATGCATTCGAATCCGGATCATTAAGAAGTGTTGATGAA CATTTGG3′

The resulting products containing the TCR β constant region sequence andhuIL15N72D gene were used as templates to generate a gene fragment byPCR using with front primer:

(SEQ ID NO: 10) 5″CTGGTGGGTTAACGGGAAGGAGGTGCACAGTGGGGTC3′and the back primer:

(SEQ ID NO: 20) 5′CCTCATGCATTCGAATCCGGATCATTAAGAAGTGTTGATGAA CATTTGG3′

The c149scTCR PCR product was digested with Pac I and Hpa I and theTCRβc/huIL15N72D PCR product was digested with Hpa I and BstB I. Thedigested gene fragments were ligated into a CMV promoter-containingpMSGV retrovirus vector. The resulting vector was designated asc149scTCR/IL15N72D-pMSGVn or pMSGV-c149IL15N72D (FIG. 8). The sequencesof the c149scTCR/huIL15N72D gene and protein are shown at FIG. 9A andFIG. 9B and FIG. 10A, FIG. 10B, and FIG. 10C, respectively.

Example 2 Generation of Transfected Host Cell Lines Producing FusionProteins

The expression vectors can be introduced into a variety of host celllines by several different transformation, transfection or transductionmethods. In one such method, CHO-K1 cells (5×10⁵) were seeded in a6-well plate and cultured overnight in a CO₂ incubator. The cells weretransfected with 5 μg of expression vector containing thec264scTCR/huIL15N72D fusion genes using 10 μL of Mirus TransIT-LT1reagent (Mirus) according to the manufacturer's protocol. The cells wereselected with 4 mg/mL of G418 (Invitrogen) one day after thetransfection. The G418 resistant cells were expanded and TCR/IL15 fusionprotein expressing cells were subcloned three times by the limitingdilution and production cell lines were screened based on the level ofsoluble fusion protein secreted into the culture media by TCR andhuIL15-specific ELISA with a capture antibody, anti-human TCR Cβantibody (BF1), and a detection antibody, biotinylated anti-human IL-15antibody (BAM 247, R&D Systems) described previously (see WO2008143794).The c264scTCR/IL15N72D producing cell line was then transducted with thepseudotyped retroviral vector containing c264scTCR/huIL15RαSushi-huIgG1fusion gene as follows.

To produce the pseudotyped retroviral vector, 2×10⁶ of the 293GPpackaging cells in a poly-lysine coated 10 cm dish (BD Bioscience) werecultured for 2 days at 37° C. in a CO₂ incubator. The cells were thenco-transfected using Lipofectamine 2000 (Invitrogen) with 9 μg of theplasmid pMC-c264scTCR/huIL15RαSushi/huIgG1 and 4 μg of the plasmid pMD-Gencoding VSV-G envelope protein. The supernatant containing virus wascollected 48 hrs post-transfection and cell debris was removed bypassing through a 0.45 μM polyvinylidene fluoride filter. Virus wasapplied to the c264scTCR/IL15N72D producing cells (1×10⁵ cells/well in a6-well plate) in the presence of 10 μg/ml of polybrene (Sigma-Aldrich).Cells were selected with 10 μg/ml of puromycin and 2 mg/ml of G418 2days post-transduction. The puromycin and G418 resistant cells wereexpanded and the T2 fusion protein complex expressing cells weresubcloned three times by the limiting dilution and production cell lineswere screened based on the level of soluble fusion protein secreted intothe culture media using a huIgG1/huIL15-specific ELISA with a captureantibody, anti-human IgG antibody (Jackson ImmunoResearch), and adetection antibody, biotinylated anti-human IL-15 antibody (BAM 247, R&DSystems).

Example 3 Generation and Purification of T2 Fusion Proteins

Cell lines expressing c264scTCR/huIL15N72D andc264scTCR/huIL15RαSushi/huIgG1 were cultured under growth conditions(i.e. 25-37° C. for 5 to 28 days in small scale culture flasks, spinneror shaker flasks or in large scale hollow-fiber, wave bag or stir tankbioreactors or equivalent culture vessels and reactors) to produce theT2 molecule as a soluble protein in the culture media. To purify the T2molecule the culture media was pH-adjusted and loaded on to animmunoaffinity column containing an anti-TCR antibody (BF1) covalentlycoupled to Sepharose. The column was washed and T2 molecules eluted with0.5 M Na-citrate pH 4.0. The eluted protein was concentrated and bufferexchanged into phosphate buffered saline (PBS) and then loaded onrProtein A-Sepharose column. Following wash steps, the protein waseluted with 0.5 M Na-citrate pH 4.0 and then buffer exchanged into PBS.The resulting protein was characterized by Coomassie-stained SDS-PAGEand size exclusion chromatography.

Under reducing SDS-PAGE conditions, the purified T2 protein migrated astwo polypeptide bands corresponding to the molecular weights expected ofthe c264scTCR/huIL15N72D and c264scTCR/huIL15RαSushi/huIgG1 componentscompared to purified c264scTCR/huIgG1 and c264scTCR/huIgG1ΔCH1 fusionproteins which migrate single bands expected of homodimeric molecules(FIG. 11). Under non-reducing denaturing conditions, thec264scTCR/huIL15RαSushi/huIgG1 band migrates at a molecular weightconsistent with a dimeric polypeptide whereas the c264scTCR/huIL15N72Dband is consistent with its monomeric form (FIG. 11). By size exclusiongel filtration chromatography, the native T2 protein eluted at theexpected molecular weight of a four-chain (2× c264scTCR/IL15N72D,2×c264scTCR/huIL15RαSushi/huIgG1) molecule (FIG. 12). These resultsconfirm that the T2 molecule exhibits a multichain conformationconsistent with the interactions between the huIL15N72D andhuIL15RαSushi domains and covalent interactions between the huIgG1 asshown in FIG. 1.

Similar mammalian cell expression and affinity chromatographypurification methods were used to generate other T2 protein complexesdescribed herein.

Example 4 In Vitro Characterization of the Binding Activities of the T2Molecule

In vitro assays were carried out to characterize the binding activitiesof the domains of the T2 molecule and to compare these activities withthose of other fusion molecules. To characterize the IgG1 domain,microtiter wells were coated with anti-human IgG1 antibody andequivalent molar amounts of purified T2 protein, composed ofc264scTCR/huIL15N72D and c264scTCR/huIL15RαSushi/huIgG1 chains, orpurified c264scTCR/huIgG1 fusion protein were applied to the wells.Following binding and washing steps, the bound proteins were detectedwith anti-human IgG1 antibody under standard ELISA conditions.

The results of the assay shown in FIG. 13 demonstrate that the IgG1domain of the T2 molecule shows equivalent antibody binding activity asthe comparable domain of the TCR/IgG1 fusion, indicating that the T2IgG1 domain retains a native conformation. The TCR domain of the T2molecule was assessed in a similar assay. Equivalent molar amounts of T2or c264scTCR/huIgG1 proteins were captured on anti-human IgG1 Ab coatedwells and detected with an anti-human TCR Cβ antibody (W4F).

As shown in FIG. 14, the T2 protein exhibited 2-fold higher reactivitythan the c264scTCR/huIgG1 protein to the anti-TCR antibody. This isexpected given the four-chain TCR fusion protein composition of the T2molecule compared with the homodimeric composition of thec264scTCR/huIgG1 fusion. The peptide/MHC binding activity of the TCRdomains of the T2 molecule was assessed. Equivalent molar amounts of T2(composed of c264scTCR/huIL15N72D and c264scTCR/huIL15RαSushi/huIgG1chains) or c264scTCR/huIgG1 proteins were captured on anti-human IgG1 Abcoated wells and detected with p53 (aa 264-272) peptide/HLA-A2streptavidin-HRP tetramers. As shown in FIG. 15, the T2 proteinexhibited 3-fold higher binding activity than the c264scTCR/huIgG1protein to the peptide/MHC reagent. This was unexpected since based onits structure and anti-TCR Ab reactivity (see FIG. 14) the T2 proteinwas anticipated to only exhibit 2-fold higher TCR binding activity thanc264scTCR/huIgG1. Thus the T2 molecular structure provides a betterantigen-specific binding activity than expected based on the individualcomponents. This enhanced binding activity may be the result of lesssteric interference, better avidity effects, cooperative interactionsand/or a better conformational fit between the TCR domain andpeptide/MHC antigen.

Example 5 Characterization of the Biological Activity of the T2 IL-15Domain

The activity of the IL-15 domain of the T2 molecule was also assessed.Microtiter wells were coated with anti-human IL-15 antibody andequivalent molar amounts of purified T2 protein, composed ofc264scTCR/huIL15N72D and c264scTCR/huIL15RαSushi/huIgG1 chains, orpurified c264scTCR/huIL15N72D fusion protein were applied to the wells.Following binding and washing steps, the bound proteins were detectedwith anti-human IL-15 antibody under standard ELISA conditions.

As shown in FIG. 16, the T2 protein exhibited increased reactivity(1.6-fold higher) compared to c264scTCR/huIL15N72D fusion for theanti-IL15 Ab, as expected based on hypothesis that each T2 moleculecontains two IL-15 domains. The biological activity of the IL-15 domainof the T2 molecules was further characterized in proliferation assaysusing the cytokine-dependent 32Dβ cell line. To measure cellproliferation, 32Dβcells (2×10⁴ cells/well) were incubated withincreasing concentrations of T2 protein (composed ofc264scTCR/huIL15N72D and c264scTCR/huIL15RαSushi/huIgG1 chains) orc264scTCR/huIL15N72D fusion protein for 48 h at 37° C. Cellproliferation reagent WST-1 (Roche Applied Science) was added during thelast 4 h of cell growth according to the manufacturer's procedures.Conversion of WST-1 to the colored formazan dye by metabolically activecells was determined through absorbance measurements at 440 nm.

As shown in FIG. 17, the T2 protein exhibits 3-fold better biologicalactivity than the c264scTCR/huIL15N72D fusion protein. This wasunexpected since based on its structure and anti-IL-15 Ab reactivity(see FIG. 16), the T2 protein was anticipated to only exhibit 2-foldhigher IL-15 activity than c264scTCR/huIL15N72D. Together these resultsillustrate a number of advantages to the T2 molecular format inproviding increased TCR binding activity and IL-15 biological activitythan was not observed with these components alone or in the context ofother fusion protein formats.

The ability of the T2 protein to promote proliferation ofIL-15-responsive immune cells was examined in a primate model.Cynomolgus monkeys (n=2, 1 m, 1 f) were injected intravenously withpurified T2 protein (composed of c264scTCR/huIL15N72D andc264scTCR/huIL15RαSushi/huIgG1 chains) at 0.5 mg/kg. Blood collected 5days later was stained for CD8 memory T cells markers (CD8 and CD95) andNK cell markers (CD56 and CD16) and compared to blood taken prior totreatment. As shown in FIG. 18, T2 treatment resulted in an expansion ofCD8⁺CD95⁺ memory T cells (A) and CD56^(dim)CD16⁺ effector NK cells (B).These results are consistent with the T2 molecule displaying potentIL-15 activity in vivo.

Example 6 Characterization of the Binding and Biological Activity of theT2 Fc Domain

The binding activity of the IgG1 Fc domain of the T2 molecule wascharacterized in cell binding assays. Fc-gamma receptor bearing U937cells were incubated with 33 nM of T2 protein (composed ofc264scTCR/huIL15N72D and c264scTCR/huIL15RαSushi/huIgG1 chains),c264scTCR/huIgG1 or A2AL9scTCR/IgG1 (negative control) for 20 min. Cellswere washed once and incubated with PE-conjugated p53 (aa 264-272)peptide/HLA-A2 tetramer for 20 min. The binding to Fc gamma receptors onU937 cell surface was analyzed with flow cytometry as shown in FIG. 19A.Similar U937 binding studies using a range of protein concentrations wasalso carried out and the mean fluorescent intensity for the stainedcells was plotted in FIG. 19B.

The results of these studies indicate that the U937 cells are stainedmore effectively with the T2 molecules than the correspondingc264scTCR/huIgG1 fusion proteins, verifying the Fc receptor bindingactivity of the T2 molecules. To assess the biological activity of theFc domains, the ability of the T2 molecule to mediate antibody dependentcellular cytotoxicity (ADCC) activity was assessed. In this study, T2protein, c264scTCR/huIgG1 or A2AL9scTCR/IgG1 (negative control) wereadded to a 96-well plate at 0.137 to 100 nM. HLA-A2-positive T2 targetcells were pulsed with 10 μM of p53 aa264-272 peptide and labeled with50 ug/ml of Calcein-AM. The fusion proteins were mixed with 1×10⁴ of thetarget cell per well and 1×10⁶/well of fresh human PBMC were added. Theplate was incubated at 37° C. in a CO₂ incubator for 2 hrs and 100 μl ofthe conditional medium were collected and analyzed for Calcein releasedfrom lysed cells. Calcein was quantitated with a fluorescence reader atEx-485 nm, Em-538 nm, and Cutoff-530 nm. The specific cell lysis iscalculated with the following formula: SpecificLysis=[exp-(background-auto release)]/[Complete release-(background-autorelease)]×100%. Exp=fusion protein+T2 cells+PBMC; Background=mediumonly; Auto release=T2 cells only; Complete release=T2 cells+0.5% TritonX-100.

The results of triplicate determinations per data point are shown inFIG. 20 where two different lots of the T2 proteins were characterized.The results indicate that the T2 protein was more effective at mediatingADCC-like activity against peptide/MHC presenting target cells than theTCR-IgG1 fusion protein. The improved activity may have been the resultof enhanced binding of the T2 molecules to the peptide/MHC complexand/or increase reactivity to the effector cells displaying Fc receptorsor IL-15 receptors.

Example 7 Characterization of T2 Molecule Binding to Peptide/MHCComplexes Displayed on Cells

To assess the binding activity of T2 protein to peptide/MHC targets oncells, HLA-A2-positive T2 cells were pulsed with various amounts of p53aa264-272 peptide. The cells were then incubated with T2 protein(composed of c264scTCR/huIL15N72D and c264scTCR/huIL15RαSushi/huIgG1chains), c264scTCR/huIgG1 or A2AL9scTCR/IgG1 (negative control), each at83 nM. The cells were incubated with biotinylated anti-TCR Ab (BF1) andstreptavidin-PE. The cell were then analyzed by flow cytometry as shownin FIG. 21A. The mean fluorescent intensity for the stained cells wasplotted in FIG. 21B.

The results show that the T2 molecules exhibit enhanced ability todetect p53 peptide/HLA-A2 complexes on cells compared to thec264scTCR/huIgG1 fusion protein. These results indicate that the T2protein is capable of binding more effectively than c264scTCR/huIgG1fusions to tumor-associated peptide antigens on target cells.

Similar results are expected using T2 molecules comprising TCR domainsspecific to other peptide/MHC targets. For example, various peptidesderived from the human tumor associated proteins; p53, gp100, MART1,MAGE-A3, PSMA, PSA, Her2/neu, hTERT, tyrosinase, survivin, WT1, PR1,NY-ESO1, EGFR, BRAF and others, are known to bind HLA molecules and betargets for human T cell responses via TCR interactions. Additionally,TCRs specific to HLA complexes displaying viral peptide antigens fromHIV, HCV, HBC, CMV, HTLV, HPV, EBV and other virus have been identified.These TCR could be fused to the IL-15 or huIL15RαSushi proteins andcharacterized for peptide/MHC reactivity on the appropriate peptideloaded antigen presenting cells as described above.

Example 8 Characterization of T2 Molecules Bearing Two Different TCRDomains

As indicated above, it is useful to have multiple different TCR domainsfused to the IL-15, IL-15Rα and IgG components of the T2 molecule. Thisallows more than one antigen targeting activity to be present in asingle multichain protein. To demonstrate the feasibility of thisapproach, c264scTCR-Sushi-hIgG1-pMSGVc and c149scTCR-hIL15N72D-pMSGVnexpression vectors were co-transfected into CHO cells cultured inIMDM-10 medium. The culture supernatant was harvested after 6 daysculture of the transfectants at room temperature. The T2 molecules ofc149scTCR/huIL15N72D and c264scTCR/huIL15RαSushi/huIgG1 werecharacterized with ELISAs. The purified T2-molecules ofc264scTCR/huIL15RαSushi/huIgG1 and c264 scTCR/huIL15N72D were used as acontrol. In one assay to assess the TCR domains, wells were coated withanti-human TCR Ab (BF1), the fusion protein was added and the boundprotein was detected with biotinylated anti-human TCR Ab (W4F-BN).

The results shown in FIG. 22 indicate that the TCR domains of T2molecules composed of c149scTCR/huIL15N72D andc264scTCR/huIL15RαSushi/huIgG1 were detectable by anti-TCR antibodies.To assess the IgG1 and IL-15 domains of the T2 proteins, an ELISAcomprised of a goat anti-human IgG Ab capture and anti-human IL-15 Abdetection described above as used.

As shown in FIG. 23, the T2 molecule composed of c149scTCR/huIL15N72Dand c264scTCR/huIL15RαSushi/huIgG1 was detectable in this formatindicating interaction between the protein chains containing the IgG andIL-15N72D domains. The activity of the c149scTCR domain was alsoexamined in an ELISA using anti-human IgG Ab capture and detection withp53 (aa 149-157) peptide/HLA-A2 streptavidin-HRP tetramers.

Shown in FIG. 24, the T2 molecule composed of c149scTCR/huIL15N72D andc264scTCR/huIL15RαSushi/huIgG1 was detectable in this format indicatingmolecules with a IgG1 domain also have binding activity to the p53 (aa149-157) peptide/HLA-A2 complex via interactions between thec149scTCR/huIL15N72D and c264scTCR/huIL15RαSushi/huIgG1 chains.Additional assays consisting of anti-human IgG Ab capture and detectionwith either p53 (aa 149-157) peptide/HLA-A2 or p53 (aa 264-272)peptide/HLA-A2 tetramers or anti-TCR Ab (BF1) capture and anti-TCR Ab oranti IL15 Ab detection verified that each of the domains wasfunctionally linked in the T2 protein composed of thec264scTCR/huIL15RαSushi/huIgG1 and c149scTCR/huIL15N72D chains (FIG.24).

T2 molecules in which these two TCR domains were expressed on the otherprotein chains, i.e. c264scTCR/huIL15N72D andc149scTCR/huIL15RαSushi/huIgG1 chains, were also generated. The Fc andTCR activity of these molecules were assessed following binding to U937cells and detection with p53 (aa 264-272) peptide/HLA-A2 tetramersfollowed by flow cytometry.

As shown in FIG. 25, T2 molecules composed of c264scTCR/huIL15N72D andc149scTCR/huIL15RαSushi/huIgG1 chains were capable of binding Fc gammareceptors on U937 cells via the Fc domain and recognizing p53 (aa264-272) peptide/HLA-A2 complex via the c264scTCR domain. These studiesverify the T2 molecules with multiple functional TCR domains and IL-15and IL15Rα and IgG1 domains are capable of forming structures as shownin FIG. 1.

Example 9 Characterization of T2 Protein Pharmacokinetics in Mice andCynomolgus Monkeys

A major limitation with potential therapies with IL-15 is the very shortbiological half-life of the cytokine in vivo. To assess the biologicalpharmacokinetic properties of the T2 molecules in an animal model,HLA-A2/Kb-transgenic mice (5 mice/timepoint) were injected intravenouslywith purified T2 protein (composed of c264scTCR/huIL15N72D andc264scTCR/huIL15RαSushi/huIgG1 chains) at 135 μg/mouse. TheHLA-A2/Kb-transgenic mouse model was selected since presence of HLA-A2.1domain, for which this c264scTCR is restricted, may influence thepharmacokinetics of the protein and should give a more relevant“humanized” view of pharmacokinetics than other non-human models. Inthis study, blood was collected at 0, 1, 4, 8, 24, 48, and 72, 96 hourspost injection and the levels of T2 protein in the serum was measured byELISA. Two different ELISA formats were used: 1) goat anti-human IgG Abcapture and anti-human TCR Ab (W4F-BN) detection or 2) goat anti-humanIgG Ab capture and anti-human IL-15 Ab detection. These assays allowassessment of the stability of the intact protein and multichain proteincomplex.

As shown in FIG. 26A, the T2 molecule had a biological half-life ofabout 9-11 hours following intravenous injection. This is considerablylonger than the reported-1 hour half-life of human IL-15 observed inmice after IP injection (Stoklasek T A et al. 2006. J. Immunol. 177:6072). Additionally the T2 molecule reached serum concentrationsconsistent with the dose delivered, whereas very little of theadministered dose of IL-15 was recovered in the serum in the studyreported previously (Stoklasek T A et al. 2006. J. Immunol. 177: 6072).Thus, the T2 molecule has a significantly better pharmacokinetic profilethan free human IL-15. In addition, based on the similar PK profileobserved with the two ELISAs, the T2 protein remained intact as amultichain molecule with no evidence of cleavage.

To assess the biological pharmacokinetic properties of the T2 moleculesin a primate model, cynomolgus monkeys (n=2, 1 m, 1 f) were injectedintravenously with purified T2 protein (composed of c264scTCR/huIL15N72Dand c264scTCR/huIL15RαSushi/huIgG1 chains) at 0.5 mg/kg. In this study,blood was collected at 0, 1, 4, 8, 24, 48, 72, 96 and 120 hours postinjection and the levels of T2 protein in the serum was measured byELISA. Three different ELISA formats were used: 1) anti-human TCR Ab(βF-1) capture and HRP conjugated goat anti-human IgG Ab detection or 2)anti-human IL-15 Ab capture and HRP conjugated goat anti-human IgG Abdetection or 3) anti-human IL-15 Ab capture and anti-human TCR Ab(W4F-BN) detection. These assays allow assessment of the stability ofthe intact protein and the multichain protein complex.

As shown in FIG. 26B, the T2 molecule had a biological half-life ofabout 4-6 hours following intravenous injection. This is considerablylonger than the reported ˜1 hour half-life of IL-15 observed in monkeysfollowing subcutaneous injection (Villinger, F. et al. 2004. Vaccine 22:3510). Thus, the T2 molecule appears to have a significantly betterpharmacokinetic profile than free IL-15. In addition, based on thesimilar PK profile observed with the three ELISAs, these data supportsthe murine PK data that suggests the T2 protein remains intact as amultichain molecule with no evidence of cleavage.

Example 10 Anti-Tumor Activity of T2 Molecules Against Solid HumanTumors in Xenograft Tumor Mouse Model

To determine the therapeutic effects of the T2 protein, we examinedantitumor activity in a primary tumor growth model with the human p53+HLA-A2+ A375 melanoma cell line in nude mice. Tumor cells were injectedsubcutaneously into nude mice and tumors were allowed to grow to 100 mm³before treatment began. Tumor-bearing mice were injected intravenouslywith 32m/dose (1.6 mg/kg) T2 protein composed of c264scTCR/huIL15N72Dand c264scTCR/huIL15RαSushi/huIgG1 chains, 32 μg/dose (1.6 mg/kg)c264scTCR/huIL2, or 60 μg/dose (3 mg/kg) 264scTCR/huIgG1. The mice weretreated every other day for one week (3 injections) followed by a 9 dayrest period and then every other day for an additional week (3injections). During the study, tumor growth was measured and the tumorvolumes were plotted (FIG. 27). The results were compared to A375 tumorgrowth in mice treated with only PBS.

As shown in FIG. 27, A375 tumor growth was inhibited in nude micetreated with either T2 molecule or TCR-IL2 or TCR-IgG fusion proteins.Previous studies showed that the antitumor effects of the p53 specificTCR-IL2 or TCR-IgG fusion proteins in this model were the results oftargeting the effector domain activity to the tumor site via the TCRdomain (Belmont et al. 2006 Clin. Immunol. 121:29, Mosquera et al. 2005J. Immunol. 174:4781). To assess this possibility, T2 proteins withnon-targeted TCR domains will be tested in the A375 tumor xenograftmouse model. A decrease in efficacy of the non-targeted T2 moleculescompared with the p53-specific T2 proteins against the A375 tumor willprovide evidence that tumor antigen targeting play a role in theantitumor activity of the T2 molecules.

Example 11 Characterization of T2 Molecules with Mutations in the IL-15and Fc Domains

As disclosed in WO2008143794, mutations can be introduced into the IL-15domain that increase or decrease its ability to interact with theIL-15Rβγ chains and affect its biological activities. For example, asindicated above, the N72D substitution can increase the IL-15 biologicalactivity 5 to 10 fold. In other instances, it is useful to decreaseIL-15 activity to provide antagonist function. To examine the effects ofsuch mutations in the context of the T2 molecular format,c264scTCR/huIL15 constructs containing substitutions at positions 8(i.e., D8N) and 65 (i.e., N65D) of the IL-15 domain were generated andco-expressed with the c264scTCR/huIL15RαSushi/huIgG1 protein. Theresulting complexes of c264scTCR/huIL15 variant andc264scTCR/huIL15RαSushi/huIgG1 chains were tested for IL-15 biologicalactivity using the 32Dβ cells as described in Example 5. As shown inFIG. 28, the T2 molecules comprising IL-15 D8N and N65D variantsexhibited a significant decrease in their ability to support 32D β cellproliferation compared to the T2 molecules comprising IL-15 N72D domainor the c264scTCR/huIL15 fusions. Consistent with the results of Example5, the T2 molecules comprising IL-15 N72D domain exhibited more IL-15activity than either the c264scTCR/huIL15N72D or c264scTCR/huIL15fusions.

Mutations were also introduced into the IgG1 Fc domain that werepreviously shown to decrease its ability to interact with Fc gammareceptor or complement (Hessell, A. J., et al. 2007. Nature 449:101-1040, incorporated herein by reference). For example, thesubstitution of leucine residues at positions 234 and 235 of the IgG1C_(H)2 (numbering based on antibody consensus sequence) (i.e. . . . P EL L GG . . . ) (SEQ ID NO: 21) with alanine residues (i.e. . . . P EA AGG . . . ) (SEQ ID NO: 22) results in a loss of Fc gamma receptorbinding whereas the substitution of the lysine residue at position 322of the IgG1 C_(H)2 (numbering based on antibody consensus sequence)(i.e. . . . K C K S L . . . ) (SEQ ID NO: 23) with an alanine residue(i.e. . . . K CA SL . . . ) (SEQ ID NO: 24) results in a loss ofcomplement activation (Hessell, A. J., et al. 2007. Nature 449:101-1040, incorporated herein by reference). These substitutions wereintroduced into the c264scTCR/huIL15RαSushi/huIgG1 construct and theresulting protein was co-expressed with c264scTCR/huIL15N72D or theother TCR-IL-15 variants described above. The ability of these complexesto mediate ADCC activity of human PBMCs against p53 aa264-272peptide-loaded HLA-A2-positive T2 target cells was assessed as describedin Example 6. Other mutations known to alter Fc function are provided,for example, in Lazar et al., PNAS, 103:4005-4010, 2006 (incorporatedherein by reference).

As show in FIG. 29, the T2 complex comprising thec264scTCR/huIL15RαSushi/huIgG1-LALA and c264scTCR/huIL15N72D chains wasnot capable of mediating high levels of ADCC activity consistent withthe loss of Fc gamma receptor binding exhibited by the Fc-LALA variant.In contrast, complexes comprising c264scTCR/huIL15RαSushi/huIgG1-KA andc264scTCR/huIL15N72D chains or the IL-15 variants (N65D or D8N)described above exhibited the same level of ADCC activity as thec264scTCR/huIL15RαSushi/huIgG1-c264scTCR/huIL15N72D complex. Withoutbeing bound by mechanism, these data are also expected based on thelikelihood that the IL-15 domain and the Fc complement-binding domainare not involved in mediating ADCC activity.

The effects of the IL-15 and Fc mutations on the ability of the T2molecules to stimulate human NK and T cell responses were also examined.Human PBMCs at 1.8 to 5×10⁵ cells/mL were incubated for 4 days at 37° C.in media containing 1 nM T2 molecules comprising the mutations describedabove or with 10 ng/mL recombinant human IL-2 or IL-15 as a control.

NK cell cytotoxicity was then assessed using NK-sensitive K-562 cells astarget cells following labeling with 50 ug/ml of Calcein-AM. Variousratios of PBMCs and K-562 cells were mixed and incubated at 37° C. in aCO₂ incubator for 2 hrs and 100 μl of the conditional medium werecollected and analyzed for Calcein released from lysed cells. Calceinwas quantitated with a fluorescence reader at Ex-485 nm, Em-538 nm, andCutoff-530 nm. The specific cell lysis is calculated with the followingformula: Specific Lysis=[exp-(background-auto release)]/[Completerelease-(background-auto release)]×100%. Exp=K−562 cells+PBMC;Background=medium only; Auto release=K-562 cells only; Completerelease=K−562 cells+0.5% Triton X-100.

As shown in FIG. 30, incubation with the T2 molecule comprising thec264scTCR/huIL15RαSushi/huIgG1 and c264scTCR/huIL15N72D chains wascapable of stimulating NK cell cytolytic activity of human PBMCscompared to that observed following incubation with media alone. Inaddition the T2 molecules comprising the Fc domain LALA and KA variantswere also capable of stimulating NK cell activity whereas thosecomprising N65D or D8N substitutions in the IL-15 domain should littleor no ability to stimulate NK cell cytotoxicity. Consistent with theseresults, incubation of human PBMCs with T2 molecules comprising thec264scTCR/huIL15RαSushi/huIgG1 and c264scTCR/huIL15N72D chains or thosewith the Fc domain LALA and KA variants resulted in an increase inproliferation of CD56+ NK cells whereas T2 molecules comprising IL-15N65D or D8N substitutions did not provide as much NK cell proliferativeactivity (FIG. 31). These results are expected based on thefunctionality of each of the IL-15 domain.

For some applications, decreased interactions between the T2 moleculesand the IL-15 or Fc receptors may be desirable to reduce non-specificbinding to cells bearing these receptors. To assess this, T2 moleculescontaining IL-15 and Fc mutations were evaluated for TCR-specific targetcell recognition using T2 cells loaded with peptide. Cell staining withthe T2 molecules or c264scTCR-streptavidin tetramer positive control wasperformed on T2 cells with (T2.265) and without loaded p53 peptide (T2)using the method described in Example 7 (FIG. 32A). Based on thestaining of unloaded cells, it is clear that the T2 molecule comprisingthe c264scTCR/huIL15RαSushi/huIgG1 and c264scTCR/huIL15N72D chains showssignificant cell binding compared to the c264scTCR-streptavidin tetrameror BF1 antibody controls. Introduction of the Fc LALA or IL-15 N65D orD8N mutations reduced this cell binding indicating that interactionswith both Fc and IL-15 receptors play a role in T2 complex binding.Combination of the Fc LALA and IL-15 N65D or D8N mutations furtherreduced T2 complex binding such that the molecule comprisingc264scTCR/huIL15RαSushi/huIgG1-LALA and c264scTCR/huIL15 D8N did notshow binding to unloaded T2 cells above the BF1 antibody negativecontrol. Staining of p53 peptide loaded cells was also effected byintroduction of the Fc or IL-15 mutations. However, when the meanfluorescence intensity of T2 molecule staining for peptide loaded versesnon-loaded cells was compared (specific to nonspecific ratio), it isclear that the T2 molecule comprisingc264scTCR/huIL15RαSushi/huIgG1-LALA and c264scTCR/huIL15 D8N chainsprovided the highest staining specificity for the p53 peptide antigen(FIG. 32B). These results indicate that the binding activities of eachof the TCR, IL-15 and IgG Fc domains of the T2 molecule can be readilyand independently manipulated to provide a multispecific complex withthe desired biological activity.

In other cases, it is useful to modify the activity of the IL-15 domainand the IgG Fc domains to optimize the therapeutic index and minimizetoxicity of the T2 complex. For example, targeted complexes relying inpart on ADCC activity for their therapeutic effect may require dosing athigh levels (i.e., 1-20 mg/kg) that exceed the tolerable dose level ofthe IL-15 component. In such a case, complexes containing a mutation inthe IL-15 domain that reduces its activity are expected to providebetter therapeutic activity and lower toxicity. T2 molecules containingN65D or D8N substitutions in the IL-15 domain described above or othersubstitutions including I6S, DBA, D61A, N65A, N72R, V104P or Q108A,which has been found to reduce IL-15 activity, are of particularinterest.

Example 12 Characterization of Non-Targeted T2 Molecules

In some applications, it is not necessary to target specific antigenswith the T2 complex. In such molecules the antigen-specific domains suchas the TCR binding domains can be inactivated by mutations or completelydeleted. Using the methods described herein, the activity of such amolecule comprising huIL15RαSushi/huIgG1 and huIL15 D8N chains referredto as T2MΔTCR was compared to the T2 molecule comprisingc264scTCR/huIL15RαSushi/huIgG1 and c264scTCR/huIL15N72D chains (referredto as T2M) and a T2 molecule lacking the huIgG1 chain(c264scTCR/huIL15RαSushi and c264scTCR/huIL15N72D, referred to as T2MΔIgor c264scTCR dimer). When tested for ability to support 32Dβ cell growthas described in Example 5, the T2MΔTCR exhibited very potent IL-15activity (FIG. 33A) that was >24 fold that observed with recombinanthuman IL-15.

The ability of the T2MΔTCR to support human immune cell growth was alsoassessed. Human PBMC at 1×10⁶ cells/ml were incubated with media in thepresence or absence of T2M (0.5 nM), T2MΔTCR (0.5 nM), or T2MΔIg (1 nM)for 7 days. Cells were stained with anti-CD45RO and anti-CD8, oranti-CD8, anti-CD95, and anti-CCR7, or anti-CD56 and anti-CD16, andanalyzed with FACScan. The averaged results from 8 different donorsshown in FIG. 33B indicate that the T2MΔTCR and other T2 molecules couldeffectively stimulate expansion of various CD8+ memory T cell and NKcell subsets including effector memory T cells. The NK cell activity ofthese cells was examined using the methods described in Example 11.Representative results from 2 donor PBMC preparations shown in FIG. 33Cindicate that the T2MΔTCR and other T2 molecules could effectivelystimulate NK cell cytolytic activity. Overall these results indicatethat the T2MΔTCR protein is a potent immunostimulatory molecule.

Example 13 In Vivo Activity of T2 Molecules

To further characterize the immunostimulatory activity of the T2molecules, T2M, T2MΔTCR, T2MΔTCR lacking the IgG1 CH1 domain(T2MΔTCRΔCH1), T2M with the Fc-LALA mutation (T2MLALA) and T2 with theIL-15 D8N mutation (T2MD8N) were tested for their ability to induceexpansion of NK and CD8 T-cells in C57BL/6 mice. In addition,c264scTCR/huIL15N72D, c264scTCR/huIL15RαSushi andc264scTCR/huIL15N72D+c264scTCR/huIL15RαSushi complexes were evaluated.

Mice were i.v. injected on day 1 and 4 with the fusion proteins at anamount equivalent to a 2.5 μg dose of IL-15. On day 8, blood cells andsplenocytes were collected, stained for CD8 T-cells and NK cells, andanalyzed by flow cytometry. The results shown in FIG. 34 indicate thatT2 molecules are effective at expanding both blood and splenic NK cellsand CD8 T cells in vivo. T2MLALA showed similar activity as T2M,suggesting FcR binding and signaling may not play a significant role inNK and CD8 T cell expansion. T2MD8N treatment resulted in decreasedactivity when compared with T2M, confirming the finding that D8Nmutation diminished the molecule's immunostimulatory activity in vitrousing human PBMC. Deletion of TCR (T2MΔTCR) and deletions of TCR and CH1(T2MΔTCRΔCH1) showed decreased activity. These effects may have been dueto the shorter half-lives of these smaller molecules. Thec264scTCR/huIL15N72D, c264scTCR/huIL15RαSushi andc264scTCR/huIL15N72D+c264scTCR/huIL15RαSushi complexes also showedreduced in vivo activity relative to the T2M, verifying the in vitroresults indicating that the T2 molecule is a more potentimmunostimulatory compound.

Example 14 Multispecific T2 Molecules

To further characterize the ability of the IL-15 and IL-15Rα/IgG Fcfusion domains to act as a scaffold for multiple binding domains, afusion protein complex (OT1-CD8-T2M) was created comprising asingle-chain TCR domain (OT1scTCR) specific for H-2K^(b)-restricted OVAaa257-264 peptide (SIINFEKL) (SEQ ID NO: 25) linked to huIL15N72D and asingle chain CD8α/β domain linked to the huIL15RαSushi/huIgG1 fusion.The single chain CD8α/β domain comprises the extracellular domain ofmurine CD8α linked via a (G₄S)₄ (SEQ ID NO: 26) peptide linker to theextracellular domain of murine CD8β. It is well characterized that CD8binds to a site in the MHC molecule distal to the TCR-specificpeptide/MHC interface. Thus both the OT1scTCR and scCD8α/β domains ofthe OT1-CD8-T2M complex are expected to interact at different sites onthe OVA aa257-264/H-2K^(b)-molecule.

To test this, binding activity of OT1-CD8-T2M was compared to that ofthe OT1scTCR/huIL15N72D fusion by ELISA. Equal molar amounts of eachprotein was captured on a well coated with anti-TCR Cβ mAb (H57) andprobed with OVA aa257-264/H-2K^(b) tetramers or mAbs to IL15, CD8α, CD8βor TCR Vα2. Assays were also performed with wells coated with anti-humanIg and probed with anti-TCR Vα2.

As shown in FIG. 35A, the OT1-CD8-T2M protein exhibited reactivity toanti-IL15, CD8α, CD8β, TCR Vα2 and human Ig antibodies. There was abouta 3-fold higher reactivity to anti-TCR Vα2 mAb than OT1scTCR/huIL15N72D,as expected based on the multivalent format of the T2M fusion complex.However, the OT1scTCR/huIL15N72D fusion showed little or no binding toOVA aa257-264/H-2K^(b) tetramers whereas binding was clearly apparentwith the OT1-CD8-T2M protein (FIG. 35B). These results indicate thatboth the OTscTCR and scCD8α/β domains of the OT1-CD8-T2M complex bind tothe OVA aa257-264/H-2K^(b) molecule to provide high affinity stableinteractions.

Example 15 IL-15:IL-15Rα Domains as a Functional Scaffold

Preparation of Peptide/MHC Class I (pMHCI) Tetramers—The murine H-2Kbgene was cloned from total RNA extracted from C57BL/6 mouse lymphocytesas described above. The extracellular region was ligated into theHLA-A*0201 heavy chain expression vector (31) replacing the HLA-A*0201coding sequence (31). The β2m, HLA-A*0201 and H-2Kb expression vectorswere individually transformed into E. coli and expression of therecombinant proteins were induced as described (31), and were expressedas insoluble inclusion bodies. The active and soluble proteins incomplex with the peptides were obtained by the re-folding methoddescribed athttp://www.microbiology.emory.edu/altman/jdaWebSite_v3/ptetPrepOverview.shtml.The p53 (aa264-272) and (aa149-157) peptide/HLA-A*0201 reagents arereferred to as A2/p53.264-272 and A2/p53.149-157, respectively, and theOVA (aa257-264) peptide/H-2Kb is referred to as Kb/OVA.257-264.

ELISA—Immunoplates (Maxisorb, Nunc, Rochester, N.Y.) were coated with(BF1) 8A3.31 mAb for capturing c264scTCR fusion proteins or with H57-597mAb for capturing OT1scTCR fusion proteins. After washing, the proteinswere detected using various probes as detailed in the Results section.ABTS (2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammoniumsalt) substrate was then added and absorbance was measured at 405 nmusing a 96-well plate reader (Versamax, Sunnyvale, Calif.).

Flow Cytometry—For characterization of the c264scTCR fusion proteincomplexes, T2 cells were pulsed with p53 (aa264-272) peptide at 37° C.for 2 h in the presence of peptide loading enhancer (PLE, AltorBioScience Corp., Miramar, Fla.). For the OT1scTCR fusion proteincomplexes, murine lymphoma EL4 cells were pulsed with OVA peptide at 100μg/ml and PLE at 37° C. for 6 h. The various birA fusion proteins(complexed with SA-PE) were added and incubated at 4° C. for 1 h. Thesamples were washed two times and analyzed on a FACScan flow cytometerusing CellQuest software (BD Biosciences, San Jose, Calif.).

To assess IL-15 domain binding activity, 32Dβ cells were incubated with320 nM of the c264scTCR fusion protein complexes for 30 min at 4° C. Thebinding of the proteins was in turn detected with biotinylated (BF1)8A3.31 mAb for 15 min and SA-PE (5 μg/ml each) for 15 min. The stainedcells were analyzed by flow cytometry as described above.

Cell proliferation assays—Cell proliferation was measured as previouslydescribed (25). Briefly, 32Dβ cells (1×10⁵ cells/well) were incubatedwith increasing concentrations of scTCR/hIL-15 or scTCR/hIL-15 muteinsin the presence or absence of an equal molar concentration ofscTCR/hIL-15RαSu for 48 h at 37° C. Cell proliferation reagent WST-1(Roche Applied Science, Indianapolis, Ind.) was added during the last 4h of cell growth according to the manufacturer's procedures. Conversionof WST-1 to the colored formazan dye by metabolically active cells wasdetermined through absorbance measurements at 440 nm. The EC₅₀ wasdetermined with the dose-response curve generated from the experimentaldata by nonlinear regression variable slope curve-fitting with Prizm4software (GraphPad Software, La Jolla, Calif.).

Surface Plasmon Resonance—The affinity constants of the OT1scTCR fusionproteins to their cognate pMHCI were determined using surface plasmonresonance (SPR) methodology on a BIAcore 2000 instrument (GE Healthcare,Piscataway, N.J.). Biotinylated pMHCI complexes were immobilized ontothe streptavidin-coated surface of a SA5 sensor chip (GE Healthcare,Piscataway, N.J.) by injecting protein at 2 μg/ml in HBS buffer (10 mMHEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% P20 surfactant, pH 7.4) at aflow rate of 10 μl/min. This resulted in 1000-1200 RU of immobilizedpMHCI complexes.

The purified OT1scTCR fusion proteins were diluted to 1 μM, 0.5 μM and0.25 μM in HBS. Each concentration was injected once (50 μl) at a flowrate of 10 μl/min over a freshly immobilized pMHCI surface as well asover a control streptavidin surface blocked with biotin (baseline) andthe binding curves were registered. The dissociation constant (K_(D))and association (k_(on)) and dissociation (k_(off)) rates werecalculated from the corrected binding curves (baseline subtracted) usingthe BIAevaluation 4.1.1 software (GE Healthcare Sciences, Piscataway,N.J.).

Creation of scTCR dimers using the hIL-15:hIL-15Rα scaffold—We havepreviously shown that a biologically active, bifunctional fusionprotein, designated as c264scTCR/hIL-15, could be created by fusing theN-terminus of hIL-15 to a three-domain, HLA-A*0201-restricted chimericTCR specific for the p53 (aa264-272) peptide antigen (c264scTCR) (25)(FIG. 36A). We constructed a similar fusion protein with c264scTCR andthe sushi-binding domain (aa 1-66) of human IL-15Rα (hIL-15RαSu), whichhas been shown to contain the structural elements responsible for hIL-15binding. This fusion protein was genetically linked to a birA peptidetag to allow for biotinylation and subsequent multimerization in thepresence of streptavidin (32). This fusion protein is designatedc264scTCR/hIL-15RαSu/birA and its expression and purification from CHOcells were similar to that of c264scTCR/hIL-15 (25). These fusionproteins are readily produced at a level of mgs per liter ofcell-culture supernatants (data not shown).

Based on the high specific binding activity between the hIL-15 andhIL-15RαSu domains, we anticipated that the fusion proteins could form aheterodimeric complex. In addition, examination of the crystal structureof the human IL-15:IL15Rα complex indicated that the N-termini of thetwo proteins are at opposite ends of the complex approximately 50 Åapart (33). Hence, fusion of the scTCR domains to these regions is notexpected to block complex formation.

Initial evidence of binding between the c264scTCR/hIL-15 andc264scTCR/hIL-15RαSu/birA fusion proteins was observed in ELISAs usingthe plate-bound c264scTCR/hIL-15RαSu/birA to capture hIL-15 andc264scTCR/hIL-15 proteins (25). To further characterize the dimericc264scTCR fusion protein complexes (referred to as c264scTCR dimer),equal molar amounts of purified c264scTCR/hIL-15 andc264scTCR/hIL-15RαSu/BirA fusion proteins were mixed and allowed toassociate at room temperature for more than 10 min. The complexes andthe individual protein fusions were evaluated by size exclusionchromatography.

As shown in FIG. 36B, the major species in the purified c264scTCR/hIL-15and c264scTCR/hIL-15RαSu/BirA fusion protein preparations displayed anSEC profile consistent with monomeric proteins (molecular weight(MW)=115 and 113 kDa, respectively) whereas the mixture of the twoproteins resulted in a major peak with a molecular weight correspondingto a dimeric complex (MW>192 kDa). Thus, the appearance of the largermolecular weight species in the c264scTCR dimer preparations is evidencethat the heterodimeric complex has been generated.

The c264scTCR dimer was compared with monomeric c264scTCR/BirA proteinfor their ability to bind the TCR-specific antigen, p53(aa264-272)/HLA-A*0201. In each case, the proteins were biotinylatedwith biotin ligase followed by complexing with SA-PE (32) to generatemultimeric flow cytometry staining reagents as previously described(32). When used to stain HLA-A*0201-positive T2 cells pulsed withvarying concentrations of p53 (aa264-272) peptide, both reagentsexhibited antigen-specific binding that increased in apeptide-concentration dependent manner (FIG. 37A). However, the stainingreagents comprising the c264scTCR dimer stained up to three times betterthan the monomer-derived c264scTCR/birA counterparts (FIG. 37B). Withoutbeing bound by mechanism, these data suggest that dimerization throughIL-15:IL-15Rα interaction preserves the functional activity of thescTCRs and increases the effective affinity of scTCR fusion complex toits cognate HLA/peptide through increased avidity. Similar results wereobserved when biotinylation via the birA tag was directed to theC-terminus of the scTCR/hIL-15 of the complex (data not shown). Thisdemonstrates that the C termini of the complex are accessible toconjugation to molecular probes of significant size (MW of streptavidinis approximately 60 kDa) without interfering with either thedimerization or antigen binding domains of the fusion protein complex.

These studies were extended to examine the possibility of generatingbispecific molecules. A second scTCR (c149scTCR) was created whichrecognizes an HLA-A*0201 restricted epitope of the human p53 proteinspanning the amino acid residues of 149 to 157 (24). This scTCR wasfused to hIL-15 and the resulting protein, designated c149scTCR/hIL-15,was co-expressed in CHO cells with the c264scTCR/hIL-15αSu/birA fusion.The fusion complex observed in the supernatant of the recombinant CHOcell culture was immobilized using an anti-IL-15 antibody and probedeither by HRP-labeled p53 (aa264-272) or p53 (aa149-157)peptide/HLA-A*0201 tetramers. As shown in FIG. 37C, the anti-IL-15antibody captured fusion protein complex was able to bind both of thepeptide-loaded HLA tetramers. The result demonstrates that theindividual scTCR molecules retain functional activity when fused to thehIL-15:hIL-15RαSu scaffold and the spatial arrangement ofhIL-15:hIL-15RαSu complex does not significantly interfere with thepacking of the scTCR domains which have an individual molecular weightof approximately 40 kDa.

To demonstrate the broad utility of the hIL-15:hIL-15RαSu scaffold forprotein dimerization, we created a second dimeric scTCR fusion complexby pairing two single-chain OT1 TCRs, one fused to the N-terminus ofhIL-15 and another to the N-terminus of hIL-15RαSu/birA protein. OT1 isa well-characterized TCR recognizing an epitope of OVA protein spanningthe amino acid residues 257 to 264 in the context of murine H-2Kb (34).OT1 single-chain TCR (OT1scTCR) gene was generated and fused to thehIL-15 and OT1scTCR/hIL-15RαSu/birA constructs for recombinant CHO cellexpression. The affinity purified OT1scTCR fusion proteins were found tohave pMHCI binding activity in ELISA using anti-mouse TCR Cβ H57antibody as a capture reagent and HRP-labeled, OVA (aa257-264)peptide-loaded H-2Kb tetramer (FIG. 42). To distinguish the differencein binding activity between the OT1scTCR dimer and OT1scTCR/birAmonomer, we conducted flow cytometry analysis similar to those describedabove for the c264scTCR dimers but with H-2Kb-positive EL4 cells loadedwith OVA (aa257-264) peptide.

As shown in FIG. 38, SA-PE tetramers comprising the OT1scTCR dimerindeed stained significantly better than those comprising monomericOT1scTCR/birA fusions. We also performed surface plasmon resonanceassays to assess the binding affinity of the OT1scTCR monomer and dimeragainst the biotinylated OVA (aa257-264) peptide-loaded H2-Kb/birAcomplexes immobilized on a streptavidin sensor chip. The apparentbinding affinity (KD) of the OT1scTCR dimer to OVA peptide/H-2Kbcomplexes was estimated to be about 30 μM, whereas no binding wasobserved for the monomeric OT1scTCR/birA fusion protein (Table 1). Thesedata confirm that dimerization through hIL-15:hIL-15Rα interactionpreserves the biological activity of the scTCRs and increases theeffective affinity of the scTCR molecule to its cognate pMHCI complexesthrough increased avidity.

Creation of an OT1scTCR/scCD8 Heterodimer—Since the CD8 molecule hasbeen previously demonstrated to play a pivotal role in the interactionbetween OT1 TCR and its cognate OVA peptide/H2-Kb complex (35-37), thehIL-15:hIL-15RαSu scaffold provides an opportunity to assess whether CD8molecule enhances OT1 TCR binding affinity to OVA peptide/H-2Kbexpressed on the cell surface and under cell-free and adhesionmolecule-free conditions. To achieve this, we first created a murine CD8molecule in single-chain format (scCD8) by fusing the extracellulardomains of the α and β chains of the murine CD8 using a flexible linker.This fusion gene was fused to the hIL-15RαSu/birA construct in aretroviral expression vector. Recombinant retrovirus was then used toinfect a CHO cell line expressing the OT1scTCR/hIL-15 fusion protein.The heterodimeric fusion protein complex was purified from thesupernatant of the cultured recombinant CHO cells using the anti-TCRantibody-based affinity chromatography as described above. This purifiedprotein was subjected to ELISA using anti-TCR antibody as the capturereagent and either the biotinylated anti-mCD8α or anti-mCD8β mAbs asprobes.

As shown in FIG. 39A, the anti-TCR Ab-immobilized fusion complexcontains both the CD8α and CD8β and, thus, indicates formation of anOT1scTCR/scCD8 heterodimer. We used flow cytometry analysis to comparethe binding activity of the OT1scTCR/scCD8 heterodimer with the OT1scTCRdimer to varying amounts of OVA peptide/H-2Kb complexes displayed on thecell surface. As shown in FIG. 39B, SA-PE staining reagents comprisingthe OT1scTCR/scCD8 heterodimer could readily detect OVA peptide/H-2Kbcomplexes on EL4 cells loaded with as little as 10 ng/ml OVA peptide,whereas little or no staining was observed at this peptide concentrationwhen comparable reagents comprising the OT dimer were used. Higherbackground OT1scTCR/scCD8 heterodimer staining was observed on EL4 cellsthat were not pulsed with peptide, suggesting peptide-independentinteractions were occurring between the CD8 domain and MHC molecules onthe cell surface. Similar effects have been reported for pMHCI tetramersbinding to CD8 molecules expressed on T cells (38).

The results for peptide-specific interactions of the OT1scTCR/scCD8heterodimer were further confirmed by surface plasmon resonanceanalysis. The binding affinity (KD) of the OT1scTCR/scCD8 heterodimer toOVA peptide/H-2Kb complexes was estimated to be 2.6 μM, which issignificantly higher than the ˜30 M observed for the OT1scTCR dimer(Table 1, FIG. 43). Neither fusion protein showed any binding to controlVSV peptide/H-2Kb complexes.

The difference in specific pMHCI binding activity is surprising giventhat the bivalent nature of the OT1scTCR dimer is expected to provideincreased functional affinity in this assay format. Additionally,similar SPR binding studies conducted with soluble TCR, CD8 α/β andpMHCI proteins as independent components showed only weak interactions(KD 30-100 μM) between CD8 protein and peptide/H-2Kb complexes and noapparent cooperative effects of CD8 on TCR:peptide/H-2Kb interactions(39-41). Taken together, these data indicate that the addition of theCD8 α/β domain to the OT1scTCR fusion has a greater impact on pMHCIbinding than creation of the bivalent OT1scTCR molecule. Our resultsfurther demonstrate that the hIL-15:hIL-15RαSu scaffold could be used tocreate functional bi-specific molecules with the flexibility toaccommodate complex protein-protein interactions. In addition, we showfor the first time that a functional CD8 molecule can be constructed asa soluble single-chain molecule and demonstrate that the scCD8 domainwhen complexed with OT1scTCR in a heterodimeric molecule enhancesTCR:pMHCI interactions in cell-free conditions without the presence ofother adhesion molecules.

Creation of Functional TCR α/β Heterodimers—As indicated above, theN-termini of the hIL-15 and hIL-15Rα domains are at distal ends of thecomplex raising questions as to whether this scaffold is suitable forfusions to polypeptides of a multi-chain protein. To determine whether asoluble, biologically active, heterodimeric TCR α/β could be constructedusing the hIL-15 and hIL-15RαSu scaffold, the C-terminal ends of theextracellular OT1 TCR Vα-Cα and Vβ-Cβ domains were linked to theN-termini of hIL-15 and hIL-15RαSu/birA chains, respectively. Based onthe published α/β TCR crystal structures, the TCR Cα and Cβ C-terminalamino acids of the properly folded OT1 TCR α/β molecule are expected tobe ˜18 Å apart (42). The OT1 TCRα/hIL-15 and OT1 TCRβ/hIL-15RαSu/birAfusion genes were cloned into two separate expression vectors andco-transfected into CHO cells. The secreted fusion protein complex waspurified using anti-TCR mAb affinity chromatography as described above.When analyzed by Coomassie-stained SDS-PAGE under reducing condition,the purified protein bands migrated at 50 kDa, consistent with thecalculated monomeric MW (40 kDa) of each of the two fusion molecules(data not shown).

The purified protein was further characterized in the functional ELISA(anti-TCR Cβ mAb capture: OVA peptide/H2-Kb tetramer probe). As shown inFIG. 40A, the purified protein was found to have equivalent pMHCIbinding activity as OT1 TCR in the single-chain format. Similar resultswere observed for hIL-15:hIL-15RαSu/birA fusions to the Vα-Cα and Vβ-Cβchains of the p53-specific 264 TCR (FIG. 40B). Previous attempts toproduce soluble α/β TCR heterodimers in mammalian cells have beenlargely unsuccessful (43,44). Thus, our results suggest that the TCR αand β chains were appropriately folded through the association of thehIL-15 and hIL-15RαSu/birA domains within the transfected cells.Intriguingly, the fusion to N-termini of the hIL-15:hIL-15RαSu scaffoldis able to provide the spatial arrangement sufficient for functionallyindependent binding domains as observed with the c264scTCR/c149scTCRheterodimeric complex while retaining flexibility to permit folding ofclosely paired chains such as the α and β chains of OT1 TCR and 264 TCR.

Biological Activity of the hIL-15 Domain for the hIL-15:hIL-15RαSuFusion Complexes—The IL-15 receptor (IL-15RβγC) binding capability ofthe hIL-15:hIL-15Rα domain of the c264scTCR dimer was evaluated by flowcytometry analysis using 32Dβ cells which carries the hIL-15Rβ and themurine γC (mγC) chains. These studies were carried out using c264scTCRdimers containing the wild-type hIL-15 domain, as well as dimers withhIL-15 mutein domains previously shown to enhance (N72D) or reduce (D8N)binding to the hIL-15Rβ chain (25). Additionally we have demonstratedthat these mutations do not affect formation of the hIL-15:hIL-15RαSucomplex (25). Following incubation with the c264scTCR dimers, the 32Dβcells were stained with anti-TCR mAb to detect cell-bound fusion proteindimers. As shown in FIG. 41A, the 32Dβ cells were stained positively bythe c264scTCR dimers containing hIL-15 wild-type or hIL-15N72D domainsbut not with those containing the hIL-15D8N domain, indicating that theIL-15:hIL-15RαSu portion of the complex retains the expected IL-15RβγCbinding activity.

The hIL-15 biological activity of the fusion protein dimers were alsoexamined in cell proliferation assays using 32Dβ cells. As shown in FIG.42B, the hIL-15 wild-type domain in the monomeric (scTCR/hIL-15 fusions)or dimeric (scTCR/hIL-15:scTCR/hIL-15RαSu) fusion formats were able tosupport the growth of 32Dβ cells in a concentration-dependent manner,exhibiting half-maximal stimulation (EC₅₀) at ˜300 pM. The hIL-15N72D orD8N domains either increased or eliminated the biological activity ofthe fusion proteins, respectively, regardless whether they were presentin the monomeric or dimeric fusions. These results are consistent withthe functional activity observed for non-fusion IL-15 cytokine carryingthe N72D or D8N mutations (25). Thus, formation of the fusion proteincomplex containing two independent TCR domains does not significantlychange the biological activity of the IL-15 domain. In contrast, therewas at least a 3 fold loss of IL-15 activity for the OT1 TCRα/βheterodimer complex (data not shown), suggesting formation of theheterodimeric TCR structure inhibits, to some extent, the ability of thehIL-15 domain to interact with hIL-15RβmγC. Additionally, these resultsindicate that the hIL-15 domain can be readily manipulated to allowenhanced or reduced receptor binding and functional activity, thusproviding additional flexibility for the use of the hIL-15:hIL-15RαSuscaffold in different applications.

Example 16 Toxicity Profile and Anti-Tumor Activity of T2 Molecules inImmunocompetent Mice

To determine the further in vivo effects of the of the T2 molecules, T2Mlacking the IgG1 CH1 domain (T2MΔCH1) and the non-targeted T2MΔTCRΔCH1(Alt-803) protein complexes, we examined toxicity and antitumor activityin tumor-bearing immunocompetent

C57BL mice. B16 (5×10⁵/mouse) or EG7 (1×10⁶/mouse) murine tumor cellswere injected subcutaneously into C57BL/6NHsd mice on study day 0.Tumor-bearing mice were injected intravenously of study days 1, 4, 8 and11 with 51 or 25.5 μg/dose T2 protein (composed of c264scTCR/huIL15N72Dand c264scTCR/huIL15RαSushi/huIgG1 chains), 47.7 μg/dose T2MΔCH1(composed of c264scTCR/huIL15N72D and c264scTCR/huIL15RαSushi/huIgG1CH2-CH3 chains) (molar equivalent to 51 μg/dose T2 protein), 16.6 or 8.3μg/dose T2MΔTCRΔCH1 (Alt-803) (composed of huIL15N72D andhuIL15RαSushi/huIgG1 CH2-CH3 chains) (molar equivalent to 51 and 25.5μg/dose T2 protein, respectively), or 1.2 μg/dose rhIL-15 (molarequivalent to 25.5 μg/dose T2 protein). During the study, animal weightsand tumor volumes were measured and the results were plotted (FIGS.44A-B and 45A-B).

Treatment with the T2M, T2MΔCH1 and T2MΔTCRΔCH1 (Alt-803) proteinssignificantly inhibited B16 (FIG. 44A) and EG7 (FIG. 45A) tumor growthcompared to that observed following PBS treatment and each of the fusionprotein complexes was more efficacious than rhIL-15 administered at anequivalent molar level. Additionally, there was little of notoxicological effect of T2M, T2MΔCH1 and T2MΔTCRΔCH1 treatment asmeasured by changes in body weight of the tumor-bearing mice (FIGS. 44Band 45B). Without being bound by mechanism, these data are consistentwith the in vivo immunostimulatory activity of these molecules inimmunocompetent animals (Example 13).

Example 17 Further Characterization of the Immunostimulatory andAnti-Tumor Activity of T2M and Derivatives Thereof

To further characterize similar targeted IL-15:IL-15Rα-Fc complexes,recombinant CHO cell lines were generated that co-express thec264scTCR/huIL-15 and c264scTCR/huIL15Rα/IgG1 Fc fusion proteins. In onecase the human IgG1 domain contained the entire heavy chain constant(CH1-CH2-CH3) and in a second case the CH2-CH3 domain (i.e. ΔCH1) or Fcdomain was used, as indicated above. The protein sequence of the humanIgG1 CH2-CH3 domain or Fc domain is shown in FIG. 46. For simplicity, inthis example, the resulting c264scTCR/huIL15N72D superagonist:c264scTCR/huIL15Rα/IgG1 CH1-CH2-CH3 complex is referred to as T2molecules (T2M) and the c264scTCR/huIL15N72Dsuperagonist:c264scTCR/huIL15Rα/IgG1 CH2-CH3 complex as T2M2 (also aboveas T2MΔCH1). The advantage of these complexes is that dimerizationthrough the Fc domains and interactions between IL-15 and IL-15Rαdomains yield tetrameric targeting molecules capable of binding toIL-15Rβγ-positive cells and Fc receptor (FcR)-positive cells.Additionally the activity of each of these domains can be analyzed bymutants that reduce interactions with the cognate receptors. Followingsoluble expression by recombinant CHO cells, these complexes werepurified to homogeneity by affinity chromatography using anti-TCR CβmAb-Sepharose and Protein A Sepharose. Size exclusion chromatographyindicated that the molecules migrated at the size expected for intactcomplexes.

Similar to analysis described above, the ELISA-based methods haveconfirmed that the scTCR and IL-15 domains of T2M and T2M2 retain theirrespective binding activities. Additionally, the IgG1 domain of T2M andT2M2 retains the ability to bind Fc receptor (FcR) bearing cells,allowing specific detection with peptide/HLA tetramers with comparableactivity to that of scTCR-IgG1 fusions. T2M and T2M2 were capable ofmediating ADCC activity of human lymphocytes against target cellsdisplaying the p53 (aa264-272)/HLA-A2 complex (FIG. 47). These resultsverify that T2M and T2M2 retain the antibody-like effector functionspreviously described for the scTCR-IgG fusions. Studies with complexescontaining Fc mutations (LALA) that reduce FcR binding activitydemonstrated that a functional Fc domain was required for ADCC activity.T2M and T2M2 also supported growth of the IL-15 dependent 32Dβ cellline, though T2M2 showed about ˜3 fold less in vitro IL-15 activity thanT2M. The ability of these molecules to stimulate immune responses inmice was also assessed. Treatment of C57BL/6 mice with IL-15 (1 mg/kg)had little or no effect on white blood cell (WBC) counts, spleen weightor the NK and CD8+ T cell populations in the blood whereas treatmentwith the IL-15N72D:IL-15Rα-IgG CH2-CH3 complex (Alt-803) (at a molarequivalent IL-15 dose) resulted in splenomegaly and elevated blood CD8+T cell levels (FIGS. 48A & B), consistent with the results observedpreviously for similar IL-15:IL-15Rα-Fc complexes. Both the T2M and T2M2complexes stimulated an increase in WBC levels, spleen weight and bloodNK and CD8+ T cell populations, with the T2M2 complex showing the morepotent immunostimulatory effect at an equivalent molar dose (despiteexhibiting lower IL-15 activity on 32Dβ cells). Similar treatmentdependent effects on NK and CD8+ T cell populations were observed in thespleen. Splenocytes isolated from T2M2 and IL-15N72D/IL-15Rα-IgG complex(Alt-803) treated mice showed cytolytic activity against NK-sensitiveYAC cells (FIG. 48C). Dose response studies indicate that these effectsare observed with a single dose level as low as 0.4 mg/kg (FIG. 49A).Treatment of nude mice with T2M2 and IL-15N72D/IL-15Rα-IgG show anincrease in the percentage of NK cells in the blood and spleen 4 dayspost treatment that decreases to near baseline levels 7 days posttreatment (FIG. 49B). Taken together, these results indicate that theT2M2 complex was capable stimulating CD8+ T cell and NK cell responsesin mice with significantly higher activity than that of IL-15 and for NKcells than that of the IL-15N72D/IL-15Rα-IgG complex (Alt-803).

The antitumor activity of these complexes was further examined in thesubcutaneous A375 xenograft model in nude mice. In initial studies,administration of recombinant human IL-15, the c264scTCR-IL15 andc264scTCR-IL15N72D fusion proteins or thec264scTCR-IL15N72D/c264scTCR-IL15Rα complex showed no effect on s.c.A375 tumor xenografts compared to PBS or c264scTCR-IL15Rα fusion proteintreatment (FIG. 50A). The lack of an effect of the TCR-IL15 fusions inthis model is likely due the inability of these proteins to stimulate NKcell responses in contrast to the reported results with thec264scTCR-IL2 fusion. As show above, when T2M complexes were tested inthis model, they exhibited modest but statistically significantanti-tumor activity consistent with their ability to stimulate NK cellproliferation (FIG. 50B). However, in contrast to treatment withequivalent molar amounts of c264scTCR-IL15 fusion, the T2M dosingschedule (4 mg/kg every other day for 3 weeks) resulted in significantweight loss and two of 6 mice died after the last dose. Clinicalobservations included mouse inactivity, hunched posture, and ruddy skin.Concurrent studies of IL-15 protein complexes in other models confirmedthat repeated every other day dosing was not well tolerated and thatweekly dosing provided immune stimulation without excessive toxicity. Achange of the dosing regimen from every other day to weekly schedule,T2M2 complex, at a dose level shown to be effective at inducing NK cellproliferation, exhibited significant more potent anti-tumor activitycompared to IL-15 or PBS treatment (FIG. 50C). More importantly, thisweekly dosing regimen was also well tolerated by the tumor-bearing nudemice and immunocompetent mice.

The toxicity profiles of the scTCR-IL15 fusions and T2M complexes wereassessed concurrently with the in vivo activity studies described above.As indicated above, 3 weeks of every other day treatment with scTCR-IL15fusions was well-tolerated by tumor bearing nude mice but T2M (4 mg/kg)treatment resulted in mortality in >30% of the animals. This was furtherevaluated in HLA-A*0201/Kb-transgenic mice administered 9, 18, or 36mg/kg T2M or molar equivalent amounts of T2M2 complexes every other dayfor 1 week. At 1 week following initiation of treatment, dose and timedependent effects on body weight and clinical observations were seen.Mice receiving 36 mg/kg T2M exhibited a 20% loss in body weight comparedto a 12% decrease observed in mice treated with equivalent amounts ofT2M2. No change in body weight was observed in mice treated with ˜9mg/kg T2M or T2M2 over the 1 week period. Interestingly the highertoxicity observed with T2M did not correlate with increased immune cellactivation as the mice treated with T2M2 showed higher levels of WBCcounts and NK cell levels than T2M-treated mice. Minimal effects onmouse body weight, spleen weight and immune cells was observed followingsingle dose i.v. administration of 0.4 mg/kg T2M2. Additionallypreliminary studies in cynomolgus monkeys indicated that a single 0.5mg/kg i.v. dose of T2M did not cause any observed toxicological effectbut was capable of inducing CD8+ memory T cell and effector NK cellexpansion. The results of these studies indicate that targeted IL-15fusion complexes can be generated that have potent immunostimulatory andanticancer activity and favorable toxicity and pharmacokinetic profiles.Through these studies an optimized TCR-targeted T2M2 (also referred toas T2MΔCH1 composed of c264scTCR/huIL15N72D andc264scTCR/huIL15RαSushi/huIgG1 CH2-CH3 chains) was defined andcharacterized. The nucleic acid and protein sequences of thec264scTCR/huIL15RαSushi/huIgG1 CH2-CH3 construct are shown in FIG. 51A,FIG. 51B, FIG. 51C, FIG. 51D and FIG. 52, respectively.

Example 18 Characterization of T2 Molecules Comprising AntibodyTargeting Domains

To demonstrate the utility of the huIL-15:huIL-15RαSu scaffold to createadditional disease targeted molecules, constructs were made linking theC-terminal end of an anti-human CD20 single chain antibody to theN-termini of huIL-15N72D and huIL-15RαSu/huIgG1 CH2-CH3 (Fc) chains. Theanti-human CD20 single chain antibody (anti-CD20 scAb) sequencecomprises the coding regions of the heavy and light chain V domains ofthe rituximab antibody linked via a flexible linker sequence. Thenucleic acid and protein sequences of the anti-CD20 scAb/hIL-15N72Dconstruct are shown in FIG. 53A and FIG. 53B and FIG. 54, respectively.The nucleic acid and protein sequences of the anti-CD20scAb/huIL-15RαSu/huIgG1 Fc construct are shown in FIG. 55A, FIG. 55B,and FIG. 55C and FIG. 56, respectively. These sequences were cloned intoexpression vectors as described above and the expression vectorstransfected into CHO cells. Co-expression of the two constructs allowedformation and secretion of a soluble anti-CD20scAb/huIL-15N72D:anti-CD20 scAb/huIL-15RαSu/huIgG1 Fc complex (referredto as anti-CD20 scAb T2M) which was purified from the CHO cell culturesupernatant using Protein A affinity chromatography.

Similar to analysis described above, the ELISA-based methods haveconfirmed formation of the anti-CD20 scAb/huIL-15N72D:anti-CD20scAb/huIL-15RαSu/huIgG1 Fc complex. Additionally, IL-15 receptor bindingand cell proliferation assays using 32Dβ cells as described aboveindicated that the complex exhibited IL-15 binding and biologicactivity. The anti-CD20 scAb T2M complex was then tested for antigenspecific binding activity against the human CD20⁺ Burkitt lymphoma Daudicell line. Daudi cells were incubated with anti-CD20 scAb T2M, c264scTCRT2M or PBS. Following a wash step, cell bound fusion protein complexeswere detected with PE-conjugated goat anti-human Ig antibody (GAH-Ig-PE)by flow cytometry (FIG. 57). The anti-CD20 scAb T2M complex showedsignificant binding to Daudi cells that was not observed with c264scTCRT2M or GAH-Ig-PE, indicating specific reactivity to these cells.

Studies were also conducted to determine whether the anti-CD20 scAb T2Mcomplexes were capable of killing CD20⁺ tumor cells via an ADCC-basedmechanism. Calcein-AM labeled Daudi target cells were mixed with humanPMBCs (E:T—100:1) and various concentrations of anti-CD20 scAb T2M,c264scTCR T2M (negative control) or chimeric anti-CD20 mAb (positivecontrol). After an incubation period, target cell lysis was evaluated asdescribed above. As shown in FIG. 58, the anti-CD20 scAb T2M complex washighly effective at mediating ADCC activity against CD20⁺ human lymphomacells. This was verified by similar studies examining different effectorto target cell ratios, where the anti-CD20 scAb T2M complex (at 2 nM)showed comparable activity as the chimeric anti-CD20 mAb (FIG. 59).

Based on these results, the anti-CD20 scAb T2M molecule is expected toexhibit antitumor activity against human lymphoma cells in standardxenograft tumor models (see for example, Rossi et al. Blood 2009;114:3864; Gillis et al. Blood. 2005; 105:3972; and Xuan et al. Blood2010; 115:2864-2871).

Additionally T2M constructs comprising anti-CD20 light chains and heavychain domains individually fused to the huIL-15N72D andhuIL-15RαSu/huIgG1 CH2-CH3 (Fc) chains, respectively (or visa versa),could be generated and expressed as described herein. The nucleic acidand protein sequences of two such fusion constructs are shown in FIG.60A and FIG. 60B, FIG. 61, FIG. 62A, FIG. 62B, and FIG. 62C and FIG. 63.Purified complexes comprising these fusion proteins are expected toexhibit Fc domain and IL-15 biologic activity, and CD20-specific bindingactivity, as described above. These complexes are expected to mediateADCC activity against CD20⁺ tumor cells and antitumor activity againstCD20⁺ tumor cells in vivo.

Similar T2M constructs comprising scAb or antibody recognition domainscould be readily generated with antibody sequences specific to other CDantigens, cytokines or chemokine receptors or ligands, growth factorreceptors or ligands, cell adhesion molecules, MHC/MHC-like molecules,Fc receptors, Toll-like receptors, NK receptors, TCRs, BCRs,positive/negative co-stimulatory receptors or ligands, death receptorsor ligands, tumor associated antigens, virus-encoded andbacterial-encoded antigens, and bacterial-specific. Of particularinterest are T2M with antibody domains specific to epitopes of CD3, CD4,CD19, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD40, CD44, CD51, CD52,CD70, CD74, CD80, CD152, CD147, CD221, EGFR, HER-2/neu, HER-1, HER-3,HER-4, CEA, OX40 ligand, cMet, tissue factor, Nectin-4, PSA, PSMA,EGFL7, FGFR, IL-6 receptor, IGF-1 receptor, GD2, CA-125, EpCam, deathreceptor 5 MUC1, VEGFR1, VEGFR2, PDGFR, Trail R2, folate receptor,angiopoietin-2, alphavbeta3 integrin receptor and HLA-DR antigens.Antibody domains against viral antigens from HIV, HCV, HBC, CMV, HTLV,HPV, EBV, RSV and other virus are also of interest, particularly thoserecognizing the HIV envelope spike and/or gp120 and gp41 epitopes. Suchantibody domains can be generated from sequences known in the art orisolated de novo from a variety of sources (i.e., vertebrate hosts orcells, combinatorial libraries, random synthetic libraries,computational modeling, etc.) know in the art.

Additionally, as indicated above, it is useful to increase or decreasethe activity of the IL-15 domain and the IgG Fc domains to optimize thetherapeutic index and minimize toxicity of the antibody-targeted T2complexes. Methods of modifying the activity of Fc domains are describedabove and are well characterized in the art. In such a case, complexescontaining a mutation in the IL-15 domain that reduces its activity areexpected to provide better therapeutic activity and lower toxicity.Antibody-targeted T2 molecules containing N65D or D8N substitutions inthe IL-15 domain described above or other substitutions including I6S,D8A, D61A, N65A, N72R, V104P or Q108A, which has been found to reduceIL-15 activity, are of particular interest.

Example 19 Co-expression of IL-15N72D and IL-15RαSu/Fc Fusion Gene inCHO Cells

Previous studies have shown that recombinant IL-15 is poorly expressedby mammalian cells (A. Ward et al., Protein Expr Purif 68 (2009) 42-48).However, it has been reported that intracellular complex formation withIL-15Rα prevents IL-15 degradation in the ER (C. Bergamaschi et al., JBiol Chem 283 (2008) 4189-4199). Hence, it was postulated that IL-15could be produced at a higher level if it is co-expressed with IL-15Rα.It is known that soluble IL-15Rα fragment, containing the so-called“sushi” domain (Su) at the N terminus, bears most of the structuralelements responsible for cytokine binding. Soluble IL-15RαSu (withoutits transmembrane domain) and IL-15 are able to form very stableheterodimeric complexes in solution (K_(d) of complex=100 pM (G.Bouchaud et al., J Mol Biol 382 (2008) 1-12)) and these complexes arecapable of modulating (i.e. either stimulating or blocking) immuneresponses via the IL-15Rβγ_(c) complex (E. Mortier et al., J Biol Chem281 (2006) 1612-1619; M. P. Rubinstein et al., Proc Natl Acad Sci USA103 (2006) 9166-9171; T. A. Stoklasek et al., J Immunol 177 (2006)6072-6080; G. Bouchaud et al., J Mol Biol 382 (2008) 1-12). Thus, acomplex consisting of IL-15N72D and an IL-15RαSu/Fc fusion protein waschosen for production (see FIG. 64). The IL-15RαSu domain wasgenetically fused to the human IgG1-Fc region to facilitate itspurification and dimerization via interchain disulfide bonds. Toco-express IL-15N72D and the IL-15RαSu/Fc, two individualretrovirus-based expression vectors, pMSGV-IL-15RαSu/Fc andpMSGV-IL-15N72D, were constructed and co-transfected into CHO cells. Therecombinant CHO cells were selected based on the neomycin and puromycinresistance elements provided by the two expression vectors, andindividual producing cell lines were then generated using limiteddilution cloning. A clone that is capable of producing approximately 100mg/L of IL-15N72D:IL-15RαSu/Fc complex (Alt-803), based on ELISA, in aserum-free, defined medium was identified. This result demonstrated thatIL-15 could be expressed at high levels in mammalian cells if it isco-expressed with the IL-15RαSu domain.

Example 20 Purification and Characterization of theIL-15N72D:IL-15RαSu/Fc Complex (Alt-803)

When IL-15RαSu/Fc and IL-15N72D were co-expressed and assembledintracellularly in recombinant CHO cells, four different forms ofproteins were expected in the cell culture supernatants: 1) dimericIL-15RαSu/Fc molecule fully occupied with two IL-15N72D subunits, 2)dimeric IL-15RαSu/Fc molecule partially occupied with one IL-15N72Dsubunit, 3) a small amount of free homodimeric IL-15RαSu/Fc moleculewith no IL-15 bound, and 4) free IL-15N72D. Since IL-15N72D lacks an Fcregion, a rProtein A-based affinity purification step was used toseparate the free IL-15N72D from all of the Fc-bearing fusion proteinsin the culture supernatant.

An ion exchange chromatography method was then developed to separatevarious forms of the IL-15RαSu/Fc complex. The calculated isoelectricpoint (pI) of the IL-15RαSu/Fc dimeric molecule is 8.5. As expected,this protein in 20 mM Tris-HCl, pH 8.0 solution was subsequently foundto not bind to QSFF resin. Additionally, the calculated pI of IL-15N72Dis 4.5. Therefore, it was predicted that the overall charge of thepartially occupied IL-15N72D:IL-15RαSu/Fc (i.e. dimeric IL-15RαSu/Fc+oneIL-15N72D molecule) and the fully occupied IL-15N72D:IL-15RαSu/Fc(dimeric IL-15RαSu/Fc+two IL-15N72D molecules) are different. This isconsistent with IEF gel analysis of the Protein-A-purified preparations,which showed two major groups of complexes with pIs between 5.6-6.5 and6.8-7.5 corresponding with the expected pIs of the fully occupied andpartially occupied complexes, respectively (FIG. 65A). The heterogeneityamong pI bands of each protein group is possibly due to the degree ofglycosylation and C-terminal lysine variants in the IgG1 chain. Thus,buffers with different ionic strengths were employed to separately elutethe partially occupied and fully occupied complexes from the QSFF. Using130 mM NaCl, 20 mM Tris-HCl, pH 8.0, a single protein fraction (Qstep 1) was eluted from QSSF and found to contain mainly the partiallyoccupied complex based on ELISAs determining the fractional occupancy ofthe IL-15RαSu/Fc molecule. In the subsequent step using 300 mM NaCl, 20mM Tris-HCl, pH 8.0, two protein fractions designated as Q1c and Q2cwere further eluted from the QSFF. ELISA analyses performed on thesepreparations indicated that Q1c fraction contained a mixture ofpartially occupied (10% of total) and fully occupied (90%) complexeswhereas Q2c fraction contained only the fully occupied complex (data notshown). These findings are consistent with IEF gel analysis of thepurified protein preparations (FIG. 65B). Proteins eluted from Q step 1have broad pIs ranging from 5.6 to 7.5; proteins of pIs 6.8 to 7.5representing the partially occupied complex. Fraction Q1c of Q step 2elution mainly contained protein with pIs ranging from 5.6 to 6.5 (i.e.fully occupied complex) but with small amounts of contaminant proteinwith pIs of 5.6 to 7.5. The Q2c fraction contained only proteins withpIs ranging from 5.6 to 6.5.

In SEC analysis, the purified IL-15N72D:IL-15RαSu/Fc (Alt-803) Q2cpreparation was found to elute as a single molecule with high purity(FIG. 66). The estimated molecular weight of the homodimer wasapproximately 114 kDa, which was larger than the 92 kDa molecular weightcalculated based on the deduced amino acid sequence of IL-15N72D andIL-15RαSu/Fc fusion proteins. This is likely due to the glycosylation ofthe proteins produced by mammalian cells.

In reducing SDS-PAGE (FIG. 65C), the purified IL-15N72D:IL-15RαSu/Fc(Alt-803) preparation was found to contain three proteins with molecularweights of 40 kDa, 16 kDa and 13 kDa. However, after a digestion withN-Glycosidase F, only two proteins, with molecular weights of ˜37 kDaand 13 kDa, were detected (FIG. 65D). These molecular weights closelymatch the calculated molecular weights of IL-15RαSu/Fc and IL-15 orIL-15N72D. This suggests that these two proteins were glycosylatedduring mammalian cell production and the IL-15N72D was produced in twomajor glycosylation forms with molecular weights of 13 kDa and 16 kDa.The relative abundance of these IL-15N72D species in the differentpurification fractions shown in FIG. 65C is consistent with levels ofcomplex occupancy determined by ELISA and IEF gel analysis.

The IL-15N72D and IL-15RαSu/Fc were separated in reducing SDS-PAGE andthe N-terminus amino acid sequences of these proteins were determinedusing the Edman degradation method. Approximately 15 N-terminal aminoacid sequences were obtained for IL-15RαSu/Fc and IL15N72D,respectively. The determined N-terminal amino acid sequences of theseproteins matched their amino acid sequences deduced from the codingregions of the two genes. The amino acid sequences for the two majorbands that appeared on reduced SDS-PAGE at 13 and 16 kDa were confirmedto be IL-15N72D. This sequence confirmation again provided the evidenceof glycosylation of IL-15N72D in mammalian cells.

Example 21 Pharmacokinetic Properties of the IL-15N72D:IL-15RαSu/FcComplex (Alt-803)

It has previously been reported that IL-15 and in vitro assembledIL15:IL-15Rα/Fc complex (Alt-803) had a 1 h and 20 h serum half-life,respectively, in mice when these proteins were injectedintraperitoneally (T. A. Stoklasek et al., J Immunol 177 (2006)6072-6080). To assess whether IL-15 and the co-expressed, purifiedIL-15:IL-15αSu/Fc complex behaved similarly when administeredintravenously, their pharmacokinetic parameters were determined in CD-1mice. Intravenous administration was chosen because this is likely theroute of drug delivery to be used for the IL-15:IL-15αSu-Fc complex inhumans. Female mice were injected intravenously with 1.0 mg/kgIL-15:IL-15αSu/Fc or 0.28 mg/kg IL-15 (a molar equivalent dose) andblood was collected at various time points from 15 min to 8 h for IL-15and 30 min to 72 h for IL-15N72D:IL-15αSu/Fc post injection. Serumconcentrations of IL-15N72D:IL-15αSu/Fc were evaluated using two ELISAformats, one (anti-IL-15 Ab detection) which detects the intact complexand the other (anti-human IgG Fc Ab detection) which detects only theIL-15αSu/Fc fusion protein. Concentrations of IL-15 were evaluated witha standard IL-15-specific ELISA.

The predicted fit and actual data for IL-15:IL-15αSu/Fc and IL-15following the single intravenous bolus injections are shown in FIG. 67.The estimated half-life of IL-15:IL-15aSu/Fc using anti-IL-15 Ab-basedor anti-human IgG Fc Ab-based ELISAs was about 25 or 18 h, respectively.These results indicate that the fusion protein was not cleaved and theIL-15 did not significantly disassociate from the IL-15RαSu/Fc moleculein vivo. The clearance (Cl) of IL-15:IL-15αSu/Fc ranged from 0.059 to0.051 mL/h and the volume of distribution at steady state (Vss) rangedfrom 2.1 to 1.3 mL depending on the assay format. In comparison, IL-15had an absorption half-life of 0.24 h and a terminal half-life of 0.64h. The Cl of IL-15 was 49 mL/h, and the Vss was 18.4 mL. These resultsindicate that IL-15:IL-15αSu/Fc displays a >24 fold longer terminalhalf-life and is cleared >800 fold slower than IL-15.

Example 22 In Vitro and In Vivo Biological Activities of theIL-15N72D:IL-15RαSu/Fc Complex (Alt-803)

The biological activity of the co-expressed and purifiedIL-15N72D:IL-15RαSu/Fc complex (Alt-803) was evaluated using an IL-15dependent 32Dβ cell proliferation assay. For this assay, an in vitroassembled (IVA) IL-15N72D:IL-15RαSu/Fc complex (IL-15N72D:IL-15RαSu/FcIVA) (Alt-803) was also generated by mixing IL-15N72D and IL-15RαSu/Fcat a 1:1 ratio for 30 min at 4° C. As shown in FIG. 68, theIL-15N72D:IL-15RαSu/Fc complex (Alt-803) had equivalent biologicalactivity as IL-15N72D:IL-15RαSu/Fc IVA (Alt-803) to support growth of32Dβ cells. The IL-15N72D:IL-15RαSu/Fc complex (Alt-803) exhibited anEC₅₀ of 15.61 pM and the IL-15N72D:IL-15RαSu/Fc IVA (Alt-803) displayedan EC₅₀ of 15.83 pM. This demonstrates that the co-expressedIL-15N72D:IL-15RαSu/Fc complex (Alt-803) is appropriately processedintracellularly and retains full IL-15 activity after purification.Thus, the method presented herein represents a better approach forgenerating cGMP-grade clinical material than current strategiesemploying in vitro assembly individually produced and in some casesrefolded proteins.

The IL-15N72D:IL-15RαSu/Fc complex (Alt-803) and IL-15 wt were alsocompared for their ability to induce the expansion of NK cells and CD8⁺T cells in C57BL/6 mice. As shown in FIG. 69, IL-15 wt has nosignificant effect on the expansion of NK and CD8⁺ cells four days aftera single intravenous dose of 0.28 mg/kg. In contrast, theIL-15N72D:IL-15RαSu/Fc complex (Alt-803) significantly promoted NK andCD8⁺ T cell proliferation in the blood and spleen, which led tolymphocytosis in blood and splenomegaly (FIGS. 69 and 70). Thesefindings are consistent with previous reports that IL-15:IL-15Rαcomplexes significantly increased the biological activities of IL-15 invivo (M. P. Rubinstein et al., Proc Natl Acad Sci USA 103 (2006)9166-9171; T. A. Stoklasek et al., J Immunol 177 (2006) 6072-6080; S.Dubois et al., J Immunol 180 (2008) 2099-2106; M. Epardaud et al.,Cancer Res 68 (2008) 2972-2983; A. Bessard et al., Mol Cancer Ther 8(2009) 2736-2745). This enhanced activity of the IL-15N72D:IL-15RαSu/Fccomplex is likely the result of a combination of the increased bindingactivity of the N72D mutein to the IL-15Rβγ_(c) complex (X. Zhu et al.,J Immunol 183 (2009) 3598-3607), optimized cytokine trans-presentationby the IL-15Rα chain in vivo (through the FcR receptors on dendriticcells and macrophage), the dimeric nature of the cytokine domain(increased avidity of binding to IL-15Rβγ_(c)) and its increased in vivohalf-life compared to IL-15 (25 h vs. <40 min).

In sum, the results described herein demonstrate that the IL-15N72D andIL-15RαSu/Fc genes can be co-expressed in recombinant CHO cells and afully occupied IL-15N72D:IL-15RαSu/Fc complex (Alt-803) can be highlypurified from cell culture supernatants using a simple scalablepurification method.

Example 23 Efficacy of ALT-803 in Murine Myeloma Models

To conduct efficacy studies in hematologic tumor models, highlytumorigenic myeloma lines 5T33P and MOPC-315P were developed from thewell-characterized 5T33 and MOPC-315 parental lines, respectively. Thesecells could populate the bone marrow (BM) and cause paralysis followingi.v. inoculation of syngeneic mice. Tumor development in 5T33P-bearingC57BL/6NHsd mice and MOPC-315P-bearing BALB/c mice was assessed bystaining myeloma cells in isolated BM cell preparations forintracellular 5T33P-specific IgG2b and MOPC-315P-specific IgAparaproteins. In C57BL/6NHsd mice, IgG2b paraprotein-positive myelomacell levels increased to over 20% of the total BM cells by 21 days after5T33P tumor cell inoculation (FIG. 71). A single i.v. treatment ofALT-803 (0.2 mg/kg) had a marked effect on 5T33P cells in the BM of micewith well-established tumors (14 days after tumor implantation),providing >90% reduction in BM IgG2b⁺ myeloma cells four days aftertreatment compared to controls (0.8% versus 11.0%, P≤0.02) (FIG. 72A).However, a molar equivalent dose of IL-15 was much less effective andonly reduced BM 5T33P cells by 53% compared to PBS-treated mice(P≥0.31). Dose response studies indicated that a single dose of ALT-803as low as 0.05 mg/kg was capable of reducing 90% of the BM 5T33P myelomacells (FIG. 73). Similar studies in BALB/c mice bearing well-establishedMOPC-315P tumors confirmed that treatment with ALT-803, but not IL-15,resulted in a significant decrease in BM myeloma cells compared tocontrols (P≤0.02, ALT-803 vs. PBS; P≥0.31, IL-15 vs. PBS) (FIG. 72B). Notoxicity was observed following treatment, indicating that ALT-803administration and its anti-tumor effects, which resulted in the rapidkilling of a large number of myeloma cells over a short duration, werewell tolerated by mice.

ALT-803 effects on mouse survival were also evaluated in these myelomamodels. 5T33P-bearing C57BL/6NHsd mice treated with a single 0.2 mg/kgdose of ALT-803 showed significantly increased survival when compared toPBS-treated mice, which all exhibited hind leg paralysis (survivalendpoint) between 21 to 35 days post tumor cell injection with a mediansurvival time (MST) of 25 days (P≤0.006) (FIG. 72C). Two or three weeklydoses of ALT-803 also provided a significant survival benefit in thismodel (P≤0.002, ALT-803 vs. PBS) (FIG. 72D) and in BALB/c mice bearingMOPC-315P tumors (FIG. 73).

Since ALT-803 treatment was capable of essentially curing mice bearing5T33P myeloma, these mice were evaluated to determine whether theyretain immunological memory against the tumor cells. As shown in FIG.72D, C57BL/6NHsd mice that survived initial 5T33P inoculation due toALT-803 treatment were not affected by 5T33P cell upon rechallenge 3months later, even in the absence of additional ALT-803 administration.These mice continued to survive over 190 days from the initial tumorcell inoculation. In contrast, all of the treatment-naïve miceadministered 5T33P cells on the same study day subsequently exhibitedparalysis with a MST of 29 days post tumor cell injection. Together,these results demonstrate that a short course of ALT-803 treatment hassignificantly greater anti-tumor activity against established BM myelomacells than IL-15 treatment, resulting in prolonged survival ofmyeloma-tumor bearing mice. ALT-803 was also capable of inducinglong-lasting protective immunologic memory against subsequent tumor cellrechallenge.

Example 24 CD8⁺ T Cells Mediate Efficacy of ALT-803 Against MyelomaCells

ALT-803 treatment effectively eliminated myeloma cells in vivo, it wastested whether ALT-803 had a direct effect on the viability andproliferation of 5T33P and MOPC-315P cells in vitro. Neither a decreasein cell numbers nor an increase in apoptotic cells was observedfollowing incubation of tumor cells with ALT-803 even at highconcentrations (FIG. 74). Thus, ALT-803 anti-myeloma activity in vivo islikely due to activation of immune responses rather than direct killingof tumor cells.

ALT-803 treatment is capable of significantly increasing the number ofNK and T cells in vivo (Han et. al., Cytokine, 56: 804-810, 2011). Todetermine if these immune cells were responsible for ALT-803-mediatedanti-myeloma efficacy, Ab-immunodepletion of CD8⁺ T cells and NK1.1⁺cells was performed in tumor-bearing mice prior to ALT-803 treatment.Effective depletion of these immune cell subsets could be achieved byintraperitoneal (i.p) administration of anti-CD8 and/or anti-NK1.1antibodies starting with injections 48 hours and 24 hours prior to tumorinoculation and weekly post-tumor inoculation. When ALT-803 efficacy wasexamined in 5T33P-bearing mice, it was found that CD8⁺ T-cell depletionalone or in combination with NK1.1⁺ cell depletion, but not NK1.1⁺ celldepletion alone, eliminated the anti-tumor effects of ALT-803 on BM5T33P myeloma cells (FIG. 75A). Consistent with these results,anti-tumor activity correlated with ALT-803-mediated increases in BMCD8⁺ T-cell and not NK cell levels (FIG. 75A). We also conducted immunecell depletion studies in 5T33P-bearing C57BL/6NHsd mice treated withALT-803 using survival as the efficacy endpoint. As described above,ALT-803 treatment effectively cured myeloma-bearing mice that otherwisedeveloped paralysis within 28 days (FIG. 75B). Depletion of NK1.1⁺ cellshad no effect on the anti-tumor activity of ALT-803, whereas depletionof CD8⁺ T cells or both CD8⁺ T cells and NK1.1⁺ cells significantlyreduced the ALT-803-mediated survival benefit to 5T33P-bearing mice(P<0.013). These results support our conclusion that CD8⁺ T cells, notNK1.1⁺ cells, play a major role in ALT-803-mediated activity against5T33P cells in C57BL/6NHsd mice.

Example 25 ALT-803 Induces CD8⁺CD44^(high) Memory T Cells to Expand,Up-Regulate Innate Receptors and Exhibit Non-Specific Cytotoxic Activity

It was previously shown that a single dose of ALT-803 at 0.2 mg/kg doselevel, but not IL-15, could significantly increase the CD8⁺ T cells andNK cells in naïve mice (Han et. al., Cytokine, 56: 804-810, 2011). Asshown in FIG. 76A, a single dose of ALT-803 (0.2 mg/kg) administered toeither normal or 5T33P-bearing C57BL/6NHsd mice resulted in a similar4-5 fold expansion of the CD8⁺CD44^(high) memory T cell population withlittle change in naïve T-cell levels. This is consistent withobservations by others that certain cytokines, such as IL-12, IL-18,IFN-γ or IL-15, can promote proliferation of CD8⁺CD44^(high) T cells,but not the naïve CD8⁺ T cells, in vivo (Zhang et al., Immunity, 8:591-599, 1998; Tough et al., J Immunol, 166: 6007-6011, 2001; Sprent etal., Philos Trans R Soc Lond B Biol Sci, 355: 317-322, 2000).

A recent study also showed that certain immunotherapies promoteantigen-nonspecific expansion of memory CD8⁺ T cells with innate-typecell receptors (Tietze et al., Blood, 119: 3073-3083, 2012). Unlike thememory CD8⁺ T cells stimulated by antigen-dependent TCR signaling whichup-regulate PD-1 and CD25 cell surface molecules, theimmunotherapy-mediated expanded memory CD8⁺ T cells express NKG2D,granzyme B, and possess broadly antigen-nonspecific lytic capability.Interestingly, it was found that the splenic memory CD8⁺ T cellsexpanded in vivo by ALT-803 treatment also expressed NKG2D and not CD25or PD-1 (FIG. 76B). To examine ALT-803-mediated changes in this cellpopulation, CD3⁺ enriched cells were isolated from spleens and lymphnodes of C57BL/6NHsd mice and labeled them with Celltrace™ Violet, andthen adoptively transferred these cells into syngeneic recipients. Twodays after transfer, the mice were treated with PBS or ALT-803 (0.02mg/kg or 0.2 mg/kg) and the phenotype and proliferation of theadoptively transferred cells were examined 4 days later. As shown inFIG. 76C, ALT-803 treatment resulted in a significant, dose-dependentincrease in proliferation of donor CD8⁺CD44^(high) T cells isolated fromspleens of recipient mice, whereas donor memory CD8⁺ T cells did notproliferate in PBS-treated mice. In the expanded memory CD8⁺ T-cellpopulation from 0.2 mg/kg ALT-803 treated mice, over 90% expressed NKG2Dwith increased positive staining in cells that underwent multiple roundsof proliferation. To rule out the possibility that this is due to anenormous expansion of a small population of NKG2D+ cells followingALT-803 treatment, similar adoptive transfer studies with sortedNKG2D^(neg)CD25^(neg)CD8⁺CD44^(high) T cells labeled with Celltrace™Violet were conducted. Treatment of recipient mice with 0.2 mg/kgALT-803 caused an increase in NKG2D⁺ memory CD8⁺ T cells from 0% to 13%(FIG. 76D; see gating strategy in FIG. 77A-1, FIG. 77A-2, FIG. 77B-1,and FIG. 77B-2). Thus, ALT-803 treatment not only induced theproliferation of the memory CD8⁺ T cells but also up-regulated the NKG2Dreceptor on their surface. Donor memory CD8⁺ T cell expressing CD25 alsoproliferated following ALT-803 treatment but the percentage of thesecells (˜4%) was the same in ALT-803- and PBS-treated mice, consistentwith the findings in 5T33P tumor-bearing mice.

To assess whether the induced CD8⁺ T cell responses were associated withchanges in antigen presentation potential in vivo, ALT-803 (0.2 mg/kg),LPS (12.5 μg/mouse) or poly IC (10 μg/mouse) was administered to normaland 5T33P-bearing C57BL/6NHsd mice and examined the up-regulation ofactivation/maturation markers on BM dendritic cells (DCs). ALT-803,unlike poly IC or LPS, did not increase MHC II (I-A^(b)), CD80 or CD40levels on BM DCs (FIG. 78A and FIG. 78B). Similar results were found forsplenic DCs. Thus, the rapid expansion of CD8⁺CD44^(high) memory T-cellpopulation stimulated by ALT-803 is unlikely a result of increasedantigen-specific responses, consistent with the results of othersdemonstrating antigen-independent activation of innate-type memory Tcells following immunotherapy or microbial or viral infection.

The cytotoxic activity of ALT-803-treated immune cells was also examinedin vitro. CD8⁺CD44^(high) T cells increased 5-fold in splenocytes and3-fold in CD8⁺ enriched splenic T cells from normal C57BL/6NHsd micefollowing a 3-day incubation with 0.2 μg/mL ALT-803. Similar to thefindings in vivo, up-regulation of NKG2D but not CD25 or PD-1 wasobserved on memory CD8⁺ T cells following ALT-803 incubation (FIG. 79A).The ALT-803-stimulated splenocytes and CD8⁺ enriched splenic T cellsexhibited elevated cytolytic activity against 5T33P cells (FIG. 79B) aswell as A20 lymphoma cell lines (FIG. 79C). Killing of 5T33P cells wasfurther enhanced by inclusion of ALT-803 during the cytotoxicity assay,suggesting a continued activation of immune cell anti-tumor activity byALT-803. Interestingly, 5T33P myeloma-targeted cytotoxicity ofALT-803-stimulated CD8⁺ enriched splenocytes was not affected byinclusion of an NKG2D blocking antibody, whereas this antibody reduced5T33P killing by whole splenocyte cultures (FIG. 79B). These resultssuggest that in vitro cytotoxicity of the NK cells in the wholesplenocyte cultures are dependent on NKG2D whereas that of CD8⁺ T cellsdoes not require NKG2D, but may be mediated through other innate-likeactivating receptors induced by ALT-803. The cytotoxicity of CD8⁺ Tcells was partially dependent of perforin expression since CD8⁺ T cellsobtained from perforin knock-out mice showed reduced 5T33P cell killingin this assay (FIG. 79D).

Overall, these studies indicate that ALT-803 potently inducesCD8⁺CD44^(high) T cells and up-regulates innate-cell receptor NKG2Dwithout the requirement of antigen-specific stimulation. Also, this typeof ALT-803-stimulated CD8⁺ memory T cells exhibit cytotoxic activityagainst myeloma and other tumor cells.

Example 26 Serum IFN-γ is Elevated by ALT-803 Treatment in a CD8⁺Cell-Dependent Manner and is Required for ALT-803-Mediated Efficacy

In addition to stimulating immune cells, a single dose of ALT-803 toC57BL/6NHsd mice was found to significantly increase serum IFN-γ levels(FIG. 80A). Immune-depletion studies were then carried out to identifythe immune cell type responsible for IFN-γ production after ALT-803treatment. As shown in FIG. 80A, depletion of CD8⁺ T cells, but notNK1.1⁺ cells, eliminated most of the high-level expression of serumIFN-γ, indicating that CD8⁺ T cells were the dominant source ofALT-803-induced IFN-γ. To further determine whether CD8⁺CD44^(high)memory or CD8⁺CD44^(low) naïve T cells were the primary producers ofIFN-γ after ALT-803 treatment, IFN-γ production of splenic CD8⁺ T cellsfrom ALT-803-treated mice were analyzed. Intracellular IFN-γ wasdetectable as early as 12 hours after ALT-803 treatment in theCD8⁺CD44^(high) memory T-cell population and the percentage of IFN-γproducing memory T cells continued to remain elevated for at least 48hrs after ALT-803 treatment (FIG. 80B). Significant ALT-803-mediatedinduction of intracellular IFN-γ was not observed in CD8⁺CD44^(low)naïve T cells. Thus, ALT-803 activates CD8⁺CD44^(high) memory T cells toproliferate and secrete IFN-γ via an antigen-independent pathway.

To determine whether induced IFN-γ plays a role in the anti-myelomaactivity of ALT-803, treatment effects on survival were evaluated inIFN-γ KO B6 mice bearing 5T33P cells. Similar to the findings inmyeloma-bearing C57BL/6NHsd mice following CD8⁺ T cells depletion,ALT-803 treatment provided little or no protection from mortality toIFN-γ KO mice after 5T33P cell inoculation, indicating IFN-γ is requiredfor ALT-803 efficacy (FIG. 80C). However, IFN-γ had no direct effect on5T33P cell growth or apoptosis in vitro (FIG. 74), consistent withprevious reports. These results support a mechanism where ALT-803activates IFN-γ production and cytotoxic activity of CD8⁺ memory T cellsand together these responses promote rapid elimination of myeloma cellsand prolonged survival of tumor bearing mice.

To assess whether IFN-γ is needed for ALT-803-mediated effects on CD8⁺memory T-cell responses, adoptive cell transfer studies were conductedusing donor Celltrace™ Violet-labeled CD8⁺ T cells from IFN-γ KO micetransferred into IFN-γ KO and wild-type recipient mice. As shown in FIG.81, ALT-803 treatment of IFN-γ KO or wild-type recipients inducedcomparable CD8⁺CD44^(high) memory T-cell proliferation and up-regulationof NKG2D of the adoptively-transferred cells. This indicates thatALT-803-induced CD8⁺CD44^(high) memory T cell responses were IFN-γindependent. Interestingly, CD8⁺ T cells isolated from IFN-γ KO miceexhibited less ALT-803-stimulated in vitro cytotoxic activity against5T33P cells than was observed in CD8⁺ T cells from normal C57BL/6NHsdmice. Without wishing to be bound by theory, together, these resultssuggest that while IFN-γ is not required for ALT-803-mediated activationand expansion of CD8⁺ memory T cells, it still plays a role inaugmenting the cytotoxicity of these cells against tumors via an as yetundetermined mechanism.

IL-15 and IL-15Rα are co-expressed and form a protein complex inantigen-presenting cells for trans-presentation to T and NK cells.Studies have shown that soluble IL-15:IL-15Rα complexes exhibit a 50fold better immune stimulatory activity in vivo than IL-15 alone andpotent efficacy against solid and metastatic tumors in various mousemodels; however, its activity against hematologic tumors has not beenreported. In this study, the anti-myeloma activity andmechanism-of-action of ALT-803, a protein complex consisting of an IL-15super-agonist mutant associated with a dimeric IL-15Rα/Fc fusionprotein, is described. As reported herein, a single dose of ALT-803 wasmuch more effective than IL-15 at reducing the levels ofwell-established murine 5T33P and MOPC-315P myeloma cells in the BM oftumor-bearing immunocompetent mice. ALT-803 was also found to prolongsurvival of 5T33P and MOPC-315P tumor-bearing mice and effectively cureda majority of the mice of tumors. Moreover, 5T33P-bearing mice cured byprior ALT-803 treatment were protected against subsequent 5T33Prechallenge, indicating that ALT-803-mediated the induction of longlasting anti-myeloma immune memory responses. These results areconsistent with the finding that ALT-803 exhibited significantly betteractivity compared to IL-15 in stimulating NK cell and CD8⁺ T-cellresponses in vivo (Han et. al., Cytokine, 56: 804-810, 2011). Thisenhanced immunostimulatory activity is likely the result of acombination of the increased in vivo half-life of ALT-803 compared toIL-15 (25 h vs.<40 min) and the dimeric nature of the cytokine domain inthe complex increasing its binding avidity to IL-15Rβγ_(c) (Han et. al.,Cytokine, 56: 804-810, 2011). Without wishing to be bound by theory, itis also possible that the Fc domain of the complex enablestrans-presentation of the cytokine to IL-15Rβγ_(c) receptor-bearing NKand T cells via binding to the Fc-γ receptors (FcγR) on the surface ofdendritic cells, macrophages, NK cells and other cell types. AnFcγR-binding deficient derivative of ALT-803 was recently generated tofurther evaluate the contribution of the Fc-γ domain to ALT-803-mediatedimmune stimulation.

Previous studies have shown that IL-15 and IL-15:IL-15Rα complexes canstimulate anti-tumor activity via either effector NK cells or T cells,demonstrating the remarkable capacity of IL-15 to induce differenteffector cell responses against diverse tumor types and tumormicroenvironments. In the 5T33P myeloma model reported here, treatmentwith ALT-803 resulted in an increase in CD8⁺ T-cell levels in the BM oftumor-bearing mice that correlated with the complex's ability to reduceBM 5T33P-cell burden. However, systemic depletion of CD8⁺ T cells, butnot NK1.1⁺ cells, was shown to largely eliminate the anti-tumor activityof ALT-803 on BM myeloma cells, the treatment-related survival benefitin 5T33P-bearing mice. This indicates that CD8⁺ T cells, but not NK1.1⁺cells, play a pivotal role in ALT-803 anti-myeloma activity. Thisfinding is perplexing since it was found that a single i.v. treatment ofALT-803 (0.2 mg/kg) had a marked effect on 5T33P cells in the BM of micewith well-established tumors, providing >90% reduction in BM IgG2b⁺myeloma cells four days after treatment. Such a robust and rapid onsetof immune responses is generally believed to only be associated with theinnate immune system. Additionally, a single dose of ALT-803 was capableof inducing high serum levels of IFN-γ and promoting the proliferationof CD8⁺ cells in non-tumor bearing mice shortly after treatment. Thesource of serum IFN-γ was largely from CD8⁺CD44^(high) T cells, notNK1.1⁺ cells, based on our immune-depletion analysis. Therefore, it wasquestioned whether the activation of CD8⁺ T cells and subsequentanti-tumor activity mediated by ALT-803 was antigen-dependent. Toaddress this, ALT-803 induced dendritic cell activation/maturation wasexamined. ALT-803 treatment did not up-regulate CD86, CD80, MHC-II andCD40 in splenic DCs from either tumor- or non-tumor-bearing micesuggested that ALT-803 did not promote antigen presentation at theinitial phase of the immune response. Thus, it appears unlikely thatantigen-dependent clonal expansion of naïve CD8⁺ T cells immediatelyafter ALT-803 treatment is responsible for the potent anti-myelomaactivity observed in mice bearing established 5T33P and MOPC-315Ptumors.

The proliferation of memory-phenotype (CD44^(high)) CD8⁺ T cells, butnot naïve CD8⁺ T cells, can be induced in vivo by the cytokines IL-12,IL-18 and IFN-γ, most likely via production of IL-15, or directly byIL-15. A recent study also showed that cytokine-mediated stimulationcould promote antigen-nonspecific expansion of memory CD8⁺ T cells witha unique phenotype. Unlike TCR signaling that up-regulates PD-1 and CD25surface markers on memory CD8⁺ T cells, treatment with IL-2 incombination with anti-CD40 antibody resulted in expansion of memory CD8⁺T cells that express NKG2D, granzyme B, and possess broad lyticcapabilities. These cells have been suggested to be responsible for thedramatic anti-tumor effects of this therapy in animal models. Herein,using the adoptive-cell transfer approach, ALT-803 alone could alsoinduce CD8⁺CD44^(high) memory T cells, but not naïve T cells, to acquireinnate cell receptors, such as NKG2D, without inducing PD-1, in vitroand in vivo. ALT-803 appears to act by both inducing CD8⁺ memory T cellproliferation and up-regulating NKG2D expression rather thanpreferentially expanding pre-existing CD8⁺CD44^(high) memory T cellscarrying this receptor. In vitro, the ALT-803-activated CD8⁺CD44^(high)memory T cells exhibited antigen-nonspecific and potent anti-tumoractivity against 5T33P myeloma. Due to the presence of the large numbersof the CD8⁺CD44^(high) memory T cells after ALT-803 treatment with aninnate-like phenotype and their high anti-tumor activity, it isconceivable that these cells represented the main effector cellsresponsible for mounting robust and rapid immune responses againstmyeloma in the initial phase after ALT-803 infusion.

A single dose of ALT-803 was capable of inducing high serum levels ofIFN-γ in mice. This activity appeared to be different from that inprevious studies in which monotherapy with IL-15 or single-chainIL-15:IL-15Rα complexes was shown to induce mouse immune cellproliferation, but not to affect serum IFN-γ levels. IL-15 has beenreported to elevate IFN-γ levels in vivo when co-administered withIL-12, IL-18 or other immune-stimulatory molecules via a cytokinefeedback cascade involving NK cells and macrophages. In contrast, theeffect of ALT-803 on serum IFN-γ levels was largely dependent onCD8⁺CD44^(high) memory T cells and not NK1.1⁺ cells. It has been foundthat treatment of mice with IL-15:IL-15Rα/Fc complexes similar toALT-803 can cause naïve CD8⁺ T cells to expand and acquire an activatedphenotype that includes the ability to secrete IFN-γ and mediateantigen-specific cytolytic function. These responses were dependent onMHC class I molecules, TCR avidity and were enhanced in the presence ofpeptide antigen, suggesting that IL-15:IL-15Rα/Fc complexes increase thesensitivity and responsiveness of naïve CD8⁺ T cells to endogenousantigen presentation. In contrast, ALT-803 has the unique feature ofinducing high levels of serum IFN-γ by activating CD8⁺ memory T cells inan antigen-independent fashion in vivo. Although IFN-γ has no directeffects on growth or induction of apoptosis of 5T33P tumor cells invitro as shown in this study, the loss of treatment-mediatedanti-myeloma activity in the IFN-γ KO mice bearing 5T33 tumorsdemonstrates the pivotal role of IFN-γ in the therapeutic potency ofALT-803. The effect of IFN-γ on ALT-803 anti-tumor activity isapparently via an indirect mechanism since ALT-803 did not lose itsability to induce IFN-γ-deficient CD8⁺CD44^(high) memory T cells inIFN-γ KO mice.

IFN-γ is a remarkable cytokine that orchestrates a diverse array ofcellular programs through transcriptional regulation of immunologicallyrelevant genes. IFN-γ skews the immune response toward a Th1 phenotypeby inducing T-bet, a critical transcription factor of Th1 cells, whichdirectly induces many Th1 cell-related genes, but indirectly suppressesthe Th2 cell-related genes. IFN-γ also orchestrates the trafficking ofspecific immune cells to sites of inflammation (e.g., tumor sites)through up-regulating expression of adhesion molecules (e.g., ICAM-1,VCAM-1) and chemokines (e.g., IP-10, MCP-1, MIG-1α/β, RANTES) (35-42).Thus, the loss of IFN-γ could lead to the loss of the Th1 cell-typeanti-tumor environment and the inability to up-regulate the necessarychemokine receptors and/or adhesion molecules on the ALT-803-activatedCD8⁺CD44^(high) T cells for trafficking to the tumor site. In addition,IFN-γ is a potent activator of macrophage which kill pathogens and tumorcells by producing reactive oxygen species and reactive nitrogenintermediates via induction of NADPH oxidase system and INOS. IFN-γ isalso known to repolarize the stage M2 tumor-promoting tumor-associatedmacrophages (TAMs) to M1 tumor-destroying macrophages at the tumorsites, which in turn could mount an effective immune response againsttumors (46, 47). Thus, IFN-γ secreted by ALT-803-activated memory Tcells could significantly contribute to the anti-tumor potency ofALT-803 by directly activating macrophages to enhance theirtumor-killing activities or to repolarize the TAMs for tumordestruction.

In summary, these results demonstrate the novel mechanism of action ofALT-803, an IL-15 super-agonist complex, against multiple myeloma thatacts mainly through its stimulation of CD8⁺CD44^(high) memory T cells toexpand, acquire an innate-type phenotype and secrete IFN-γ independentof antigen requirement resulting in enhancement of host survival. Thesefindings suggest a novel therapeutic strategy of exploiting theinnate-cell function of adoptive immune cells. Thus, the presentinvention not only provides for treatment of multiple myeloma, but alsofor the treatment of other cancers and infectious diseases.

Example 27 Alt-803 is Effective Against Lymphoma

ALT-803 is a fusion protein complex consisting of the IL-15N72Dsuperagonist and a dimeric IL-15 receptor alpha (IL-15Rα) sushidomain/IgG1 Fc fusion protein. Previous studies have shown thatinterleukin-15:interleukin-15 receptor alpha complex exhibits potentactivity against murine melanoma in immunocompetent mice. The studydescribed herein below is designed to test the effect of ALT-803 onprimary tumor growth of murine EG7-OVA lymphoma in immunocompetentC57BL/6 mice.

To evaluate the effect of ALT-803 when administered via intravenous(i.v.) injection in multi-dose regimen on primary subcutaneous tumorgrowth of murine EG7-OVA lymphoma in C57BL/6 mice the following studieswere carried out. Four treatment groups, ALT-803 (0.415 and 0.83 mg/kg),recombinant human interleukine-15 (rhlL-15, 0.06 mg/kg) and PBS(control), were examined in the study (Table 1). Female C57BL/6 mice(8-10 weeks old) were injected subcutaneously (s.c.) with EG7-OVA (1×106cells in 100 μl PBS) on study day (SD) 0. PBS (n=5), rhlL-15 (n=5) orALT-803 (n=5) were administered i.v. on SD 1, 4, 8, and 11 post EG7-OVAtumor cell injection. Tumors (width and length) were measured over thecourse of study. Tumor volume was the primary end point of the study.

TABLE 1 Experiment design Dosing EG7-OVA Grp Test article (mg/kg) n =Inj on ALT-803 i.v. Inj on 1 PBS — 5 SD 0 SD 1, SD 4, SD 8, SD 11 2ALT-803 0.415 5 SD 0 SD 1, SD 4, SD 8, SD 11 3 0.830 5 SD 0 SD 1, SD 4,SD 8, SD 11 4 rhIL15 0.060 5 SD 0 SD 1, SD 4, SD 8, SD 11

Female C57BL/6 mice were injected s.c. with EG7-OVA (1×10⁶cells/mouse)on study day 0. Tumor bearing mice (n=5) were treated with ALT-803 at0.415 mg/kg and 0.83 mg/kg, or rhlL-15 at 0.06 mg/kg via intravenousadministration through the lateral tail vein for a total of 4 injectionson SD 1, SD 4, SD 8 and SD 11. Mice received PBS served as a control.During the study, mouse body weight and tumor width and length weremeasured and recorded.

Four ALT-803 i.v. treatments at 0.415 mg/kg and 0.83 mg/kg significantlyinhibited tumor growth when compared with PBS control (P<0.001 andP<0.001, respectively), with Tumor Growth Inhibition (TGI) of 63.5% and68.3%, respectively, over PBS More importantly, ALT-803 treatment atboth 0.415 mg/kg and 0.83 mg/kg showed significantly better anti-tumoreffects than rhlL-15 treatment (P<0.001 and P<0.01, respectively) withTGIs of 47.1% (0.415 mg/kg ALT-803) and 54.1% (0.83 mg/kg ALT-803)compared to rhlL-15 treatment. ALT-803 treatment did not causesignificant body weight reduction, suggesting that the treatmentregimens are safe.

The anti-tumor effect of ALT-803 intravenous treatment was evaluated ina mouse lymphoma model in immunocompetent C57BL/6 mice. C57BL/6 mice(8-10 weeks old) (n=5 mice/group) were injected s.c. with EG7-OVA cells(1×106 cells/mouse) on day 0. ALT-803 (0.415 or 0.83 mg/kg), rhlL-15(0.06 mg/kg) or PBS (as a control) was administered i.v. on 1, 4, 8, and11 days post tumor cell injection. Four i.v. administrations of ALT-803significantly inhibited EG7-OVA primary tumor growth when compared withthe PBS control (P<0.001) (FIG. 82). More importantly, ALT-803 treatmentat either the 0.415 mg/kg and 0.83 mg/kg dose showed significant tumorgrowth inhibition when compared with rhlL-15 treatment (P<0.001, andP<0.01, respectively). In sum, Alt-803 significantly inhibited tumorgrowth (FIGS. 83A and 83B).

Example 28 ALT-803 Treatment Did not Significantly Affect Mouse BodyWeight

ALT-803 treatment at the dose levels utilized did not cause significantmouse body weight reduction (FIG. 84), although ALT-803 at 0.83 mg/kgshowed a transient body weight reduction after the 2nd injection. Thesedata suggest that four ALT-803 i.v. treatments at 0.83 mg/kg were safeand efficacious in inhibiting murine EG7-OVA lymphoma primary tumorgrowth in C57BL/6 mice.

Example 29 Alt-803 Inhibited HIV Infection

In the NSG mouse model, spleens were injected with human PBMC andreplication competent HIV expressing luciferase. 16×10⁶ activated PBMCswere co-injected with replication-competent HIV that contains aluciferase reporter into NSG mouse spleens. 24 hours post injection, themice were i.v. injected with either PBS (n=3) or 0.2 mg/kg of ALT-803(n=4).

One week later, the mice were sacrificed, the spleens were removed andsplenic lysates were measured for luciferase activity. FIG. 85 showsthat Alt-803 exhibited a high degree of inhibition of infection.

Example 30 CD20-targeted IL-15N72D:IL-15Rα/Fc Fusion Protein Complexes(2B8T2M) have Anti-Lymphoma Activity

As described herein, a scaffold comprising IL-15 and IL-15Rα/IgG Fcdomains (Alt-803) was developed. Using this scaffold, novelcancer-targeted immunotherapeutic agents can be generated includingthose capable of binding CD20. Specifically, a fusion protein complex(2B8T2M) with a single-chain Ab (scFv) derived from the VL-VH domains ofrituximab linked to both an IL-15 superagonist variant and an IL-15Rα/Fcfusion has been generated (FIG. 87). High affinity interactions betweenthe IL-15 and IL-15 receptor α domains and disulfide bonding between theIgG domains resulted in a stable, soluble, four-chain polypeptidecomplex that retained functional binding activity of each of itscomponents.

To create the CD20-targeted IL-15N72D:IL-15Rα/Fc fusion protein complex(2B8T2M), VL and VH gene fragments of rituximab (2B8 Ab) were cloned inan scFv format (2B8scFv), and this sequence was linked to the N-terminalcoding region of the IL-15N72D and IL-15Rα/Fc gene constructs.Expression vectors carrying these constructs were co-transfected intoCHO cells and stable cell lines with high-level production of the 2B8T2Mcomplex (i.e., >50 mg/L) were selected. The 2B8T2M complex was producedand purified. To serve as Ab controls for testing, constructs weregenerated to produce the chimeric 2B8 mAb (C2B8) equivalent to rituximabin recombinant CHO cells.

2B8T2M and C2B8 (rituximab) proteins were purified by Protein A affinitychromatography, and analyzed by SDS-PAGE and native size exclusionchromatography (SEC) (FIGS. 88A and 88B). Both purified 2B8T2M and C2B8(rituximab) proteins exhibited two polypeptide bands at the appropriateMW on SDS-PAGE (i.e. 2B8T2M comprised of 2B8scFv/IL-15N72D and2B8scFv/IL-15Rα/Fc) and eluted as a single peak for the tetramericprotein complex by SEC. The observed complex MW on SEC was less than thecalculated MW for both proteins presumably due to their structuralcharacteristics.

The 2B8T2M protein was also reactive in ELISAs with an anti-IgG Abcapture and anti-IL-15 Ab probe format. The results confirm formation ofthe stable 2B8T2M complex (FIG. 87). As a control for anti-CD20targeting activity, an IL-15N72D:IL-15Rα/Fc complex comprising scTCRdomains that bind p53 peptide/HLA-A2 complexes was used (Wong et al.,Protein Eng Des Sel, 24: 373-383, 201). This protein (264T2M) does notbind the HLA-deficient Daudi lymphoma used in our studies. Finally, aCD20-targeted IL-15N72D:IL-15Rα/Fc complex (2B8T2MLA) containing Fcmutations that reduce FcR and complement binding activity was generatedto define the role of the Fc domain in antitumor activity.

The IL-15 activity of the complexes was assessed based on proliferationof the IL-15-dependent 32Dβ cell line using the WST-1 reagent asdescribed previously (Wong et al., Protein Eng Des Sel, 24: 373-383,201). The 2B8T2M, 2B8T2MLA and 264T2M had similar IL-15 activity (FIG.89A). Binding of the complexes to the CD20-positive human Daudi lymphomacell line was assessed by flow cytometry. As expected, the 2B8T2M andC2B8 proteins showed CD20 binding with C2B8 binding slightly better than2B8T2M (presumably due to reduced activity of the scFv format) whereasthe control 264T2M complex does not bind Daudi cells (FIG. 89B). Theseresults verify the functionally of the IL-15:IL-15Rα and anti-CD20 Abdomains of the 2B8T2M complex.

Anti-CD20 Abs mediate their activity against B-cell lymphomas in partthrough complement dependent cytotoxicity (CDC), antibody-dependentcellular cytotoxicity (ADCC), and direct programmed cell death (PCD).Additionally, due to different modes of binding, type I Abs such asrituximab primarily exhibit CDC and ADCC activity whereas type II Absprimarily exhibit PCD and ADCC. These activities were compared betweenthe 2B8T2M complex and C2B8 (rituximab). 264T2M complex was used as anon-targeted IL-15 control, and 2B8T2MLA was used to assess the role ofthe Fc domain. Addition of the 2B8T2M complex and C2B8 Ab to humaneffector cells resulted in similar levels of ADCC against Daudi targetcells as assessed in a calcein release assay (FIG. 90). Little or noactivity was seen with the non-targeted 264T2M or the 2B8T2MLA Fc mutantcomplexes (FIG. 4). Both the 2B8T2M complex and C2B8 Ab were alsocapable of mediating comparable CDC activity of human serum againstDaudi cells (FIG. 91). In contrast, 264T2M was inactive and the 2B8T2MLAFc mutant showed reduced activity. The ability of the proteins todirectly induce cell death of Daudi cells was evaluated (FIG. 92). Asexpected, C2B8 Ab showed minimal activity. However, surprisingly the2B8T2M complex exhibited significant direct cell killing activityagainst Daudi cells consistent with the levels reported for type II Abs(Cragg et al., Blood, 103: 2738-2743, 2004). Daudi cell death was alsomediated by the 2B8T2MLA Fc mutant, indicating independence of Fcfunction. However, CD20 binding is clearly required based on the lack ofactivity of the 264T2M control.

Together, these studies demonstrate the 2B8T2M complex contains all ofthe properties seen in both type I and II anti-CD20 Abs, a novelcharacteristic. To evaluate the cumulative ADCC and PCD activity andimmune stimulation by IL-15, purified human T cells+NK cells wereincubated with PKH67-labelled Daudi cells (E:T 2:1) in media containingthe fusion protein complexes for 2 days. Daudi cell death was thenassessed by propidium iodide (PI) staining. All of the IL-15 complexeswere capable of inducing LAK cell activity resulting in Daudi cell death(FIG. 93). Daudi cells incubated with 2B8T2M showed even higher levelsof cell death presumably due to ADCC and PCD. This was further enhancedby CD20 targeting activity that was partially dependent on Fc activity.Overall, the antitumor activity of the 2B8T2M complex exceeded that ofthe C2B8 Ab, indicating that 2B8T2M is a superior therapeutic agentagainst CD20+ B-cell lymphomas.

In summary, the 2B8T2M complex was capable of directing ADCC and CDCagainst human lymphoma cells with comparable activity to that seen withrituximab (also referred to as C2B8 mAb). Importantly, higher levels ofdirect cell killing of human lymphomas were seen with 2B8T2M than withrituximab. Without being bound to a particular theory, this indicatesthe fusion protein complex, unlike rituximab, has both type I and typeII anti-CD20 Ab characteristics. In addition, this also indicates thatthe IL-15 superagonist/IL-15Rα components of 2B8T2M provide potentantitumor immunostimulatory activity against human B-cell lymphomas.

Without being bound to a particular theory, the 2B8T2M complex couldpotentiate anti-CD20 Ab activity by: (1) providing a molecule with bothtype I and type II characteristics, and (2) expanding the population andactivity of effector cells to augment the ADCC/phagocytic function. TheIL-15 component of 2B8T2M may also provide potent immunostimulatoryactivity to IL-15βγc receptor-bearing NK cells, macrophage and T cellsfor anti-CD20-independent tumor-killing activity (FIG. 87). The abilityto target IL-15 to the tumor microenvironment increases IL-15 activityat the tumor site and reduces potential systemic toxicities.

Example 31 CD20-targeted IL-15N72D:IL-15Rα/Fc Fusion Protein Complexes(2B8T2M) Demonstrated Antitumor Efficacy in a Well-Established HumanLymphoma Xenograft Model

As the Daudi-SCID mouse model has been used to assess the antitumoractivity of various anti-CD20 antibodies including rituximab (Cragg etal., Blood, 103: 2738-2743, 2004), the Daudi-SCID mouse model wasimplemented to further characterize the anti-CD20 scFv/IL-15:anti-CD20scFv/IL-15Rα/IgG Fc protein complex (2B8T2M) in vivo. SCID injected i.v.with 10⁷ Daudi cells developed lymphoma tumors in their bone marrowresulting in paralysis/mortality within 30 days. Daudi cells were alsoreadily detectable 14 days after i.v. injection by staining withanti-HLA-DR Ab (FIG. 94).

An efficacy study of 2B8T2M against Daudi B Lymphoma in SCID Mice wasperformed (FIG. 95). Fox Chase SCID Female mice (6 per group) wereinjected (i.v.) with 10×10⁶ human CD20+ Burkitt lymphoma Daudi cells onstudy day (SD) 0. The tumor-bearing mice were intravenously treated onSD 18 and 21 with PBS, anti-CD20 antibody (C2B8) or anti-CD20 scAb T2M(2B8T2M) at various dose levels. There was a dose dependent increase inanti-CD20 scAb T2M (T2M) levels in the serum 4 days after treatment(FIG. 96). These results are consistent with the extended half life ofthe fusion protein complex. The mice were sacrificed on SD 25 and thelevels of Daudi cell present in the bone marrow was determined based onpositive staining with PE-conjugated anti-HLA-DR antibody. Comparison ofsurvival may be analyzed using the log-rank test. Unpaired t tests withWelch's correction assuming unequal variances can be used to comparedifferences in continuous variables among the treatment groups. P<0.05(two-tailed) is defined as statistically significant.

At study day 25 post tumor cell injection, Daudi cells represented 75%of the cells of the bone marrow in mice treated with phosphate bufferedsaline (PBS) (FIG. 97). Treatment with 10 mg/kg anti-CD20 antibody(C2B8) resulted in a slight decrease in bone marrow Daudi cells whereastreatment with anti-CD20 scAb T2M (T2M) at 10 mg/kg further reduced thelevel of Daudi cells in the bone marrow to ˜25%. Anti-CD20 scAb T2M(T2M) at as low as 1 mg/kg was also effective at reducing the Daudi celllevels in the bone marrow. Anti-CD20 scAb T2M (T2M) treatment alsoresulted in a dose dependent increase in spleen weights consistent withits immunostimulatory activity (FIG. 98). Additionally, anti-CD20 scAbT2M (T2M) treatment resulted in a dose dependent increase in NK cells inthe bone marrow and spleen, consistent with its immunostimulatoryactivity (FIG. 99). NK cell percentages in the bone marrow and spleenwere unchanged following treatment with anti-CD20 antibody (C2B8).

Thus, the combinations of activities exhibited by 2B8T2M have thepotential to provide significant clinical benefit to treatment-naïve andrefractory NHL and CLL patients beyond that seen with current anti-CD20Ab-based approaches.

The above examples 1-22 were carried out using the following materialsand methods. Construction of vectors for protein complex expression

The IL-15RαSu/Fc fusion gene was constructed by overlap PCRamplification of DNA templates encoding the sushi domain of humanIL-15Rα (aa1-66 of human IL-15Rα) and the human IgG1 Fc fragment. Thesignal peptide-IL-15RαSu coding region (R. L. Wong et al., Protein EngDes Sel 24 (2011) 373-383) and human IgG1-Fc gene fragment (L. A.Mosquera et al., J Immunol 174 (2005) 4381-4388) were amplified usingthe primer pairs:

BA494: (SEQ ID NO: 16) 5′-GACTTCAAGCTTAATTAAGCCACCATGGACAGACTTACTTCTTC-3′; BA550R: (SEQ ID NO: 27)5′-GTGAGTTTTGTCACAAGATTTCGGCTCTCTAATGCATTTGA GACTGGGGGTTG-3′, andBA550F: (SEQ ID NO: 28) 5′GAGCCGAAATCTTGTGACAAAACTCAC-3′; BA393R:(SEQ ID NO: 15) 5′-GTAATATTCTAGACGCGTTCATTATTTACCAGGAGACAGGGAGAGGCTCTTC-3′,respectively. The resulting IL-15RαSu/Fc fusion gene was ligated into apuromycin-resistant expression vector pMSGV-1 (M. S. Hughes et al., HumGene Ther 16 (2005) 457-472) to construct the expression vectorpMSGV-IL-15RαSu/Fc.

The coding sequence of IL-15N72D (X. Zhu et al., J Immunol 183 (2009)3598-3607) was cloned into a modified retrovirus expression vectorpMSGV-1 (M. S. Hughes et al., Hum Gene Ther 16 (2005) 457-472) thatcarries the neomycin resistance gene after an IRES region to constructthe expression vector pMSGV-IL-15N72D.

Co-Expression of IL-15N72D:IL-15RαSu/Fc Fusion Complex in CHO Cells

To co-express IL-15N72D and IL-15RαSu/Fc fusion proteins (see FIG. 64),pMSGV-IL-15RαSu/Fc and pMSGV-IL-15N72D were co-transfected into CHOcells followed by selection in medium containing 2 mg/mL G418 (Hyclone,Logan, Utah) and 10 μg/mL of puromycin (Hyclone, Logan, Utah). TheIL-15RαSu/Fc fusion protein was also expressed individually in CHO cellsfor use in loading of recombinant human wild-type IL-15 (IL-15 wt) as acontrol. For production of the fusion proteins, the recombinant CHOcells were grown in serum free defined medium (SFM4CHO, Hyclone, Logan,Utah) at 37° C. When the viable cell density of the cultures reached amaximum, the incubation temperature was shifted down to 30° C. foraccumulation of the soluble complex. Culture supernatants were thenharvested when the viable cell density of the cultures reachedapproximately 10% viable cells.

Purification Procedure

The recombinant CHO cell culture medium was centrifuged and filtered toremove cells and debris before the supernatant was adjusted to pH 8.0with 1 M Tris-HCl, pH 8.0. The soluble IL-15N72D:IL-15RαSu/Fc fusionprotein complex (Alt-803) was purified using a two-step affinity and ionexchange chromatography-based process.

Since the IL-15N72D:IL-15RαSu/Fc complex (Alt-803) contains the IgG1-Fcdomain, an rProtein A Sepharose Fast Flow (GE Healthcare) column wasused as the first step in the purification process. Prior to sampleloading, the column was washed with 5 column volumes (CV) of 20 mMTris-HCl, pH 8.0, sanitized with 5 CV of 0.1 N NaOH for 1 h, and thenequilibrated with 7 CV of 20 mM Tris-HCl, pH 8.0. The supernatant wasloaded onto the 11 mL column at 2 mL/min, and the column was then washedwith 8 CV of 20 mM Tris-HCl, pH8.0, followed by 7 CV of washing buffer(0.1 M Na-citrate, pH 5.0) to remove non-specifically bound proteins.The protein was then eluted with 0.2 M Na-citrate, pH 4.0 and the pH ofcollected peak fractions was immediately adjusted to pH 3.5 using 0.2 Mcitric acid; the eluted protein was held at this low pH for 30 minutesas a standard viral clearance step. After the low pH hold step, the pHof the eluted preparation was adjusted to pH 7.7 by using 2 M Tris-HCl,pH 8.0. The preparation was concentrated and buffer exchanged into 20 mMTris-HCl, pH 8.0 by using an Amicon Ultra-15 centrifugal concentrator(30 kDa cut-off, Millipore, Billerica, Mass.) before sterile filtrationusing a 0.22 μm filter (Corning Life Sciences, Lowell, Mass.).

The protein preparation was then applied to a Q Sepharose Fast Flow(QSFF; GE Healthcare Bio-Sciences, Piscataway, N.J.) ion exchangecolumn. A 5 mL column was washed with buffer A (20 mM Tris-HCl, pH 8.0),sanitized by 5 CV of 0.1 N NaOH for 1 h, and then equilibrated withbuffer A. The protein concentration in the preparation was firstadjusted to <1 mg/mL with 20 mM Tris-HCl, pH 8.0 and was then loadedonto the QSFF column at a rate of 1 mL/min. The protein was then elutedfrom the column using a three-step-gradient process as follows: 20 mMTris-HCl, pH 8.0, 130 mM NaCl for four CV as the first step, 20 mMTris-HCl, pH 8.0, 300 mM NaCl for four CV for the second step and 20 mMTris-HCl, pH 8.0, 1 M NaCl for two CV as the last step. Protein peakfractions were collected, buffer exchanged into PBS (Hyclone, Logan,Utah), and filtered using a 0.22 μm filter. Protein concentration wasdetermined by UV spectrophotometer at 280 nM using an extinctioncoefficient of 1 A_(280 nm)=0.79 mg/mL. This extinction coefficient wascalculated based on the deduced amino acid sequence of theIL-15N72D:IL-15RαSu/Fc complex (Alt-803).

Individually expressed IL-15RαSu/Fc was purified using rProtein Aaffinity chromatography as described above for assembling of complex insolution with IL-15N72D or IL-15 wt produced in E. coli and refolded(Zhu, 2009 #3315). These in vitro assembled complexes were used asstandards for biological activity evaluation and estimation of degree ofoccupancy of the IL-15 binding sites in co-expressed complexes.

Gel Electrophoresis and Size Exclusion Chromatography (SEC) Analysis

Purified proteins were analyzed by different types of gelelectrophoresis methods, which included NuPAGE 12% Bis-Tris gel (underreduced and non-reduced conditions), 4-20% Tris-glycine gel (nativecondition), and IEF pH3-10 gel (for pI determination). All supplies werefrom Invitrogen (Carlsbad, Calif.). Experimental methods were performedas described by the manufacturer. Superdex 200 HR 10/30 (GE HealthcareBio-Sciences) chromatography with PBS (Hyclone, Logan, Utah) as therunning buffer was used to examine purity and to estimate molecular massof the proteins.

N-Terminal Amino Acid Sequence and Glycosylation Analysis

Protein bands of interest were separated on SDS-PAGE gels, blotted ontoPVDF membrane and stained by Ponceau S solution. N-terminal amino acidssequencing was performed using the Edman degradation method (MolecularStructure Facility, UC Davis, Davis, Calif.).

To examine whether the fusion complex was glycosylated, 50 μg of thehighly purified protein after the ion exchange chromatography wasdigested with 2 μL of N-Glycosidase F (Calbiochem, La Jolla, Calif.) ina total volume of 50 μL in PBS at room temperature for 48 h and then wassubjected to electrophoresis in NuPAGE 12% Bis-Tris gel under a reducedcondition.

Determination of IL-15N72D Occupancy of the PurifiedIL-15N72D:IL-15RαSu/Fc Complex

Purified IL-15RαSu/Fc was loaded with IL-15 wt (produced in E. coli andrefolded, provided by J. Yovandich, NCI, Fredrick, Md.) at variousratios for 15 h at 4° C. After incubation, the IL-15 wt: IL-15RαSu/Fccomplex was purified using rProtein A affinity chromatography asdescribed above. This purified complex was evaluated using two ELISAformats, one (anti-human IgG Fc capture and anti-IL-15 detection) whichdetects the intact complex and the other (anti-human IgG Fc capture andanti-human IgG Fc detection) which detects only the IL-15αSu/Fc fusionprotein. The ratio between the intact IL-15 wt: IL-15αSu/Fc complex andIL-15RαSu/Fc protein levels reflects the occupancy rate of the IL-15binding sites of the complex. [Occupancy rate (%)=the intact complex(ng/mL)/IL-15RαSu/Fc (ng/mL)×100%]. Fully occupied complex(pre-associated of IL-15RαSu/Fc and IL-15 wt at a 1:3 ratio) was thenused as a standard to quantitate the occupancy rate of purifiedIL-15N72D:IL-15RαSu/Fc fusion protein complexes (Alt-803) afterpurification.

Determination of IL-15 Biological Activity

An in vitro cell proliferation assay using the IL-15-depended 32Dβ cellline was employed to assess the IL-15 biological activities of thepurified complex and IL-15 wt proteins as previously described (X. Zhuet al., J Immunol 183 (2009) 3598-3607).

Pharmacokinetic Evaluation

The pharmacokinetic profile of IL-15N72D:IL-15RαSu/Fc complex (Alt-803)and IL-15 wt were evaluated in female CD-1 mice (4 mice/time point,Harlan, Indianapolis, Ind.) as previously described for IL-2 (H. J.Belmont et al., Clin Immunol 121 (2006) 29-39). Serum levels of theIL-15N72D:IL-15RαSu/Fc complex (Alt-803) were assessed with the twoELISA formats described above. IL-15 wt levels were assessed by ELISAusing anti-IL-15 capture (MAB647; R&D Systems, Minneapolis, Minn.) andanti-IL-15 detection (BAM247; R&D Systems, Minneapolis, Minn.).IL-15N72D:IL-15RαSu/Fc (Alt-803) levels from each ELISA format were fitwith a one-compartment model using PK Solution 2.0 (Summit ResearchServices, Montrose, Colo.). Data from mice treated with IL-15 wt werebest modeled as a two-compartment model.

Lymphocyte Stimulation

C57BL/6 mice (male, 6 wks of age, Harlan, Indianapolis, Ind.) wereinjected intravenously with a single dose of IL-15N72D:IL-15RαSu/Fcfusion complex (Alt-803) at 1 mg/kg or human IL-15 wt at 0.28 mg/kg(molar equivalent dose), respectively, or PBS as a negative control.Four days after treatment, pooled blood (5 mice per group) andsplenocytes were collected. PBMCs were isolated from the blood usinghistopaque (Sigma, St. Louis, Mo.). The PBMC and splenocytes were thenstained with PE-labeled anti-CD19, PE-labeled anti-CD335 (NKp46),FITC-labeled anti-CD4 and FITC-labeled anti-CD8 antibodies (BioLegend,San Diego, Calif.). The stained cells were analyzed on a FACScan flowcytometer (BD Bioscience, San Jose, Calif.). All animal studies wereperformed following Altor's IACUC approved protocols.

The following peptides were used in the studies presented in the aboveExamples.

Protein Amino acids Sequence SEQ ID NO: p53 149-157 STPPPGTRV 29 p53264-272 LLGRNSFEV 30 OVA 257-264 SIINFEKL 25 VSV 52-59 RGYVYQGL 31

The following protein domain linker sequences of the fusion proteinsused in the Examples presented.

Linker sequences disclosed as SEQ Linker Linker Sequences ID NO:Fusion Protein Single- TCR Vα-DTSGGGGSG 32 c264scTCR/hIL-15, chainGGGSGGGGSGGGGSSS- c264scTCR/hIL-15RαSu/birA TCR TCR Vβ linkerTCR Vα-TSGGGGSGG 33 c149scTCR/hIL15N72D GGSPGGGGSGGGGSSS- TCR VβTCR Vα-DTSGGGGSG 34 OT1scTCR/birA GGASGGGGSGGGGSSS- TCR VβTCR Vα-SGGGGSGGG 35 OT1scTCR/hIL-15D8N, ASGGGGSGGGGS-OT1scTCR/hIL-15RαSu/birA TCR Vβ Mutated TCR domain-VNEPK 36c264scTCR/hIL-15RαSu/birA, human SSDKTHTSPPSPTR-OT1scTCR/hIL-15RαSu/birA, IgG1 hIL-15RαSu OT1TCRβ/hIL-15RαSu/birA, hinge264TCRβ/hIL-15RαSu/birA TCR domain-VNEPK 36 264TCRα/hIL-15D8N,SSDKTHTSPPSPTR- OT1TCRα/hIL-15, hIL-15 OT1scTCR/hIL-15D8N BirAhIL-15RαSu-SGGGS 37 c264scTCR/hIL-15RαSu/birA, linker GGGGSID-birA tagOT1TCRβ/hIL-15RαSu/birA Single- CD8α-SGGGGSGGGGS 38scCD8αβ/hIL-15RαSu/birA chain CD8 GGGGSGGGGS-CD8β linker

Results presented in Examples 23-26 were carried out with the followingmaterials and methods.

Mice and Tumor Cell Lines

C57BL/6NHsd and BALB/c mice (5-6 week old females, Harlan Laboratories)and interferon-γ (IFN-γ) knockout (KO) [B6.129S7-Ifngtm1Ts/J] andperforin KO [C57BL/6-Prf1tm1Sdz/J] mice (5-6 week old females, TheJackson Laboratory) were housed in the animal facilities at AltorBioScience. All animal studies were performed according to NIH animalcare guidelines under IACUC approved protocols.

The murine 5T33 multiple myeloma cell line (20) was kindly provided byDr. Ulrich von Andrian, (Harvard Medical School, Boston, Mass.). Themurine MOPC-315 myeloma cell line was purchased from American TypeCulture Collection (ATCC). Tumor cell sublines, 5T33P and MOPC-315P,were developed by passage of the parental myeloma cells in C57BL/6NHsdand BALB/c mice, respectively. All cells were routinely cultured in I-10media at 37° C. with 5% CO₂ and harvested for animal injection at 80-90%confluency.

Tumor Models

Following intravenous (i.v.) injection with 1×10⁷ 5T33P cells/mouse,100% of C57BL/6NHsd mice developed tumor-induced hind leg paralysisbetween 20-30 days. Similar tumor take rates were observed in BALB/cmice following i.v. injection of 1×10⁷ MOPC-315P cells/mouse.Tumor-bearing mice were monitored daily for hind leg paralysis, signs ofovert disease progression and mortality.

ALT-803 (IL-15N72D:IL-15RαSu/Fc) was generated as described previously(Han et. al., Cytokine, 56:804-810, 2011). Recombinant human IL-15 (21)was kindly provided by Dr. Jason Yovandich (NCI, Fredrick, Md.). ALT-803at 0.2 mg/kg/dose (or as indicated), IL-15 at 0.056 mg/kg/dose (IL-15molar equivalent dose of 0.2 mg/kg ALT-803) or PBS as control wasadministered i.v. via the lateral tail vein to tumor-bearing mice.Levels of BM myeloma cells and hind leg paralysis or survival wereassessed as study endpoints.

Flow Cytometry and ELISA Analysis.

To quantitate levels of murine lymphocyte subsets, BM, spleen, lymphnode and blood were collected separately from each mouse, cells wereprepared and stained with fluor-labeled antibodies (Abs) specific toCD4, CD8, CD11c, CD19, CD25, CD40, CD44, CD80, CD107a, I-A(b), IFN-γ,IgG2b, IgA, NK1.1, NKG2D, NKp46, and/or PD-1, and appropriate isotypecontrols (eBiosciences, BD Biosciences, and Biolegend) as indicated infigure legends. Cell staining was analyzed on a FACSverse (BDBiosciences). The sorting of NKG2D^(neg)CD25^(neg)CD8⁺CD44^(high) Tcells was conducted with FACS Aria and analyzed with Diva software (BDBiosciences).

Levels of 5T33P and MOPC-316P cells in BM preparations, and IFN-γ insplenocytes were assessed by intracellular staining with Abs specific toIgG2b, IgA and IFN-γ, respectively.

IFN-γ levels in mouse serum were quantitated by ELISA using anti-IFN-γAb (AN-18) capture and biotinylated anti-IFN-γ Ab (R4-6A2) detectionfollowing the manufacturer's instruction (Biolegend).

In vivo depletion of mouse NK1.1⁺ cells and CD8⁺ T cells. For in vivodepletion of NK1.1⁺ cells and CD8⁺ T cells, mice were injectedintraperitoneally (i.p.) with 200 μg/dose anti-NK1.1 (PK136, ATCC)and/or 500 μg/dose anti-CD8 (53-6.72, ATCC) Abs. Control mice receivedPBS (0.2 mL). In pilot studies, the efficiency of NK1.1⁺ cell and CD8⁺T-cell depletion was monitored by flow cytometry following staining ofPBMCs and BM cells with appropriate Abs.

T Cell Labeling and Adoptive Transfer.

CD3⁺ enriched cells (prepared with Mouse CD3⁺ T Cell Enrichment Column,R&D System), CD8⁺ enriched T cells [positive, CD8a (Ly-2) MicroBeads,mouse, Miltenyi Biotech] or sorted NKG2D^(neg)CD25^(neg)CD8⁺CD44^(high)memory T cells from spleens and lymph nodes of donor C57BL/6NHsd orIFN-γ KO B6 mice were labeled with Celltrace™ Violet (Invitrogen) at 1.5μM/1×10⁶ cells/ml, and then 1 to 1.5×10⁶ violet labeled cells wereadoptively transferred into syngeneic C57BL/6NHsd or IFN-γ KO B6recipients on day 0 (SD0). On SD2, mice were treated (i.v.) with thefollowing test articles 0.02 mg/kg ALT803, 0.2 mg/kg ALT-803 or PBS. OnSD6, spleens were harvested and splenocytes were analyzed forproliferation of donor cells (violet label) or staining with antibodiesspecific to CD25, PD-1, CD44, CD8a, and NKG2D.

In Vitro Cytotoxicity Assay

Tumor target cells (i.e., 5T33P, A20) were labeled with PKH67(Sigma-Aldrich) according to the manufacturer's instructions. CD8⁺ Tcell enriched spleen cells from normal, IFN-γ KO, and perforin KO B6mice were isolated (untouched, CD8a⁺ T Cell Isolation Kit II, mouse,Miltenyi Biotech). Effector populations were produced by culturingprepared cells (2×10⁷) in RPMI-1640 complete media containing ALT-803(200 ng/mL) for 72 hr. Resulting effector cells were harvested, washedtwice, and re-plated into 24 well plates with PKH-labeled tumor targetcells (E:T ratio; 10:1) in media containing varying doses of ALT-803.After incubation for 20-24 hrs at 37° C. with 5% CO₂, target cellkilling was assessed by analysis of PI staining of PKH67-labeled tumorcells on a BD FACScan.

Data Analysis

Data are expressed as the mean±SE. Survival data was analyzed using thelog-rank test and Kaplan-Meier method. Comparisons of continuousvariables were done using Student's t tests or ANOVA (two-tailed)(GraphPad Prism Version 4.03). P values of less than or equal to 0.05are considered significant.

Experiments described in Example 27 were carried out as follows.

Tumor Cells

Murine EG7-OVA tumor cell line, which was derived from mouse thymoma EL4cells transfected with chicken albumin cDNA, was obtained from AmericanType Culture Collection, Manassas, Va., USA. EG7-OVA cells were culturedin RPMI 1640 medium with 1.0 mM sodium pyruvate and supplemented with0.05 mM 2-mercaptoethanol and 10% fetal bovine serum in 5% CO2 and at37° C.

Animals

Female C57BL/6 mice, 8-10 week-old, were purchased from HarlanLaboratories (Indianapolis, Ind., USA). Each mouse was identified by toeclipping. The animals were acclimated to the facility and released fromquarantine by an animal caretaker one week after arrival. Allexperimental procedures and handling mice were performed according toNIH animal care guidelines under IACUC approved protocols.

Animals were housed 5-6 animals per cage in plastic cages with bedding.The cages were placed on stainless-steel racks and identified with acage card bearing the animal identification numbers and equipped with awater bottle that provided autoclaved tap water. Autoclaved tap waterand Harlan Teklad Global 18% Protein Rodent Diet (Harlan Teklad 2918S)were available ad libitum throughout the study. The feed was analyzed bythe manufacturer for concentrations of specified heavy metals,aflatoxin, chlorinated hydrocarbons, organophosphates, and specificnutrients.

Environmental controls were set to maintain the following animal roomconditions: temperature range between 68° F. and 79° F., relativehumidity range between 30% and 70%, a minimum of 10 air changes perhour, and a controlled diurnal cycle (12-hour light/12-hour dark).Actual temperature and relative humidity in the animal room weremonitored and recorded once daily.

Reagents

ALT-803, lot #: 051910, manufactured by Altor Bioscience Corporation.

Recombinant human interleukine-15, lot #L0801006, provided by Dr. J.Yovandich, NCI (4).

Dulbecco's phosphate buffered saline (PBS)—HyQ® DPBS, Ca++- andMg++-free, Cat #SH30028FS, HyClone.

RPMI 1640 1×, Cat. #22400-089. lot #927164, GIBCO.

Fetal Bovine Serum, Cat. #SH30071.03, lot #AVB64134, HycloneLaboratories, Inc. GIBCO™ MEM Sodium Pyruvate Solution 100 mM (100×)liquid, Cat #11360070, GIBCO.

2-Mercaptoethanol, Cat #21985-023, GIBCO

Cell Culture

Mouse lymphoma tumor cells, EG7-OVA, were cultured in RPMI 1640 mediumsupplemented with 1.0 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol and10% fetal bovine serum at 37 oC with 5% CO2. The cells were washed twicewith PBS and re-suspended in PBS at 10×106 cells/mL for s.c. injection.

Subcutaneous Injection of EG7-OVA Tumor Cells

C57BL/6 mice were shaved in the rear flank before injecting tumor cells.For each animal, EG7-OVA (1×106 cells in 100 μL of PBS) was injecteds.c. on the rear flank.

Tumor Measurement

The length (mm) and width (mm) of the tumors were measured and recorded.The tumor volume was estimated using the following equation: Tumorvolume=½ (Length×Width2).

Experiment Design and Treatment Schedule

Mice injected with EG7-OVA cells were treated intravenously via thelateral vein on 1, 4, 8 and 11 days post-tumor implantation with eitherALT-803 at 0.415 or 0.83 mg/kg in 100 μL PBS or rhlL-15 at 0.06 mg/kg orPBS (100 μL) as control (Table 1). The tumor-bearing mice weremaintained to assess tumor growth rates among the treated groups.

Data Analysis

Data were analyzed by ANOVA using GraphPad Prism Version 4.03. P valuesof less than or equal to 0.05 are considered significant. Tumor growthinhibition (TGI) was calculated using the following equation: TGI(%)=(Vcontrol−Vtreated)/Vcontrol×100, where Vcontrol is the mean tumorvolume of mice from the PBS or rhlL-15 treatment control, and Vtreatedis the mean tumor volume of mice receiving test article treatment.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A kit comprising: an effective amount of aninterleukin-15N72D:IL-15 receptor alpha Sushi/Fc(IL-15N72D:IL-15RαSu/Fc) complex, wherein the complex comprises adimeric IL-15RαSu/Fc and two IL-15N72D molecules (ALT-803); anddirections for use of the kit.
 2. The kit of claim 1 wherein theIL-15RαSu/Fc comprises SEQ ID NO.:
 1. 3. The kit of claim 1, wherein aIL-15N72D molecule comprises SEQ ID NO.:
 2. 4. The kit of claim 1,wherein the effective amount is between about 1 and 20 μg/kg.
 5. The kitof claim 1, wherein the effective amount is about 1 μg/kg.
 6. The kit ofclaim 1, wherein the effective amount is about 5 μg/kg.
 7. The kit ofclaim 1, wherein the effective amount is about 10 μg/kg.
 8. The kit ofclaim 1, wherein the effective amount is about 20 μg/kg.
 9. The kit ofclaim 1, wherein the effective amount is between about 20 μg and 100μg/kg.
 10. The kit of claim 1, wherein the effective amount is about 30μg/kg.
 11. The kit of claim 1, wherein the effective amount is about 50μg/kg.
 12. The kit of claim 1, wherein the effective amount is about 75μg/kg.
 13. The kit of claim 1, wherein the effective amount is about 100μg/kg.